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Why is the number of PCR cycles limited?

Why is the number of PCR cycles limited?


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I've been told that the maximum number of cycles in PCR is between 20 and 30.

Is this true, and what are the reasons for this limitation?


I would draw the line beyond 35, but thats a bit cosmetic. The reasons are manyfold:

  • due to the exponential fashion of the amplification (ideally) reagents are used up at some point
  • reagents degrade, this is especially true for the dNTPs
  • the activity of the enzyme, despite being heat-stable is declining over time
  • beyond 35 cycles the exponential curve is flattening out (reasons see above)
  • if you run the PCR for too long, you will get more and more side-products (mostly primer dimers, but mis-aligned primers can also make problems), this is more a problem for real-time PCR than for ones run on a gel

If you need a higher sensitivity with more cycles, you can use the technique called "nested PCR". There you do a first round with a primer pair specific for the region of interest and then do a second round with primers which are located slightly to the inside of the amplified DNA. This is done to avoid the amplification of unwanted contaminations. Since you do some 50-70 rounds of PCR amplification in total, this method is extremely sensitive (also to contaminations). See the image from the Wikipedia article for details:


How many cycles of PCR before dNTPs run out?

Assume a 25 μl reaction.

Assume 200 μM dNTPs.

200 μM dNTPs = 200 pmol μl -1

so in 25 μl reaction, there are 5000 pmol of dNTPs

= 5000 x 10-12 x 6 x 1023 molecules

= 3 x 1015 molecules dNTP

Assume that we start with 1 molecule of a 1000 bp template, 50% GC

1 kb = 2000 nucleotides

So , how many of these molecules can we construct using 3 x 1015 molecules dNTP?

= 3 x 1015/2000

= 1.5 x 1012 DNA molecules

How many cycles of PCR to produce this many from a single template molecule?

2n = 1.5x 1012

nlog2 = log(1.5) + log(1012)

nlog2 = 12.18

n = 12.18/ log2 = 41 cycles

Of course this in an absolute upper limit. The estimate assumes that you start with one template molecule of 1 kb, and that dNTPs aren't being hydrolysed, or otherwise degraded.


In my own research I've found that the amount of PCR product increased as I increased the cycles to 50 times. This was for a single pair of primers used to amplify genomic DNA extracted with a particular kit as doesn't apply to other reactions I've done. What some people seem to forget is that the PCR products don't necessarily double at each cycle at any point in the PCR because some primers don't function as well as others and reaction conditions may be suboptimal (e.g. presence of contaminants or concentration of Mg ions).

In short, provided you aren't getting reduced-quality products (e.g. smearing or non-specific priming products), then I think it's fine to increase the number of cycles well beyond 30 - it's something you have to test empirically with your particular primers, template, polymerase, buffer etc.


Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is a test tube version of the same process of DNA replication that is found in the living cell. It is a fast and inexpensive way to amplify , or make many copies of, small segments of DNA. This is necessary because methods used for analyzing DNA (determining the DNA base pair sequence) require more DNA than may be in a typical sample. A particularly useful feature of PCR is that it allows the amplification process to be limited to specifically targeted segments of the DNA mixture--such as the Y chromosome markers used in genealogical testing.

When your vials of cheek cells or saliva arrive at the lab for testing, the DNA is extracted and purified. They are first mixed with a detergent which causes the cells to burst open and release their DNA along with other cell contents. The mixture is then washed with a phosphate containing buffer (mild salt) solution to dilute cellular debris. With minimal preparation, the sample is ready and the DNA on the targeted area of a chromosome can be amplified.


About those RT-PCR Tests

His case rests principally on the inappropriate use of the RT-PCR test. A colleague of mine has graciously agreed for me to reproduce his assessments of this test – he is not alone. Hat Tip Gerry @ http://boomfinanceandeconomics.com/#/

PCR is a difficult technology to grasp because there are so many subtle aspects. PCR technology has been around since 1987 when Kary Mullis invented it. He won the Nobel Prize for doing so. It is not a test that clinicians order lightly. In normal clinical medical practice, it is ordered only when dealing with very ill patients. Careful consideration and very careful interpretation are needed and the clinical situation is critical.

Everyone should watch the videos of Kary Mullis on YouTube and Bitchute. He was a genius and brilliant scientist unfortunately, he died recently — just before the Covid outbreak. https://www.bitchute.com/video/8KsH34IGgqBw/

Quote from Kary Mullis, Nobel Prize winner and inventor of PCR Tests:

“Guys like Fauci get up there and start talking, you know, he doesn’t know anything really about anything and I’d say that to his face. Nothing. The man thinks you can take a blood sample and stick it in an electron microscope and if it’s got a virus in there you’ll know it. He doesn’t understand electron microscopy and he doesn’t understand medicine and he should not be in a position like he’s in. Most of those guys up there on the top are just total administrative people and they don’t know anything about what’s going on in the body.

You know, those guys have got an agenda, which is not what we would like them to have being that we pay for them to take care of our health in some way. They’ve got a personal kind of agenda. They make up their own rules as they go. They change them when they want to. And they smugly, like Tony Fauci does not mind going on television in front of the people who pay his salary and lie directly into the camera.”

The number of PCR cycles can have dramatic consequence.

This is the kernel of the issue although there are many, many more factors. PCR tests cannot distinguish between live (viable) micro-organisms and dead ones. They both contain genetic material. This is why using PCR tests with high Ct numbers (Amplification Cycle Numbers) in a Screening situation is ridiculous. It must never be used in non-symptomatic people to “diagnose” illness.

Because of this problem, it can never be regarded as a purely “diagnostic test”. No clinicians worth their salt will ever regard a stand-alone PCR test as diagnostic. We have over 20 years of clinical experience with this technology. If you do more than 30 Amplification Cycles, you are guaranteed to find non-viable (dead) viral remnants or contaminants. Many nations have been doing 40 – 45 cycles during the Covid phenomenon. The cycle number is guaranteed to produce a high rate of positive tests that are, in fact, false. We call them “False Positives” in the world of clinical medicine.

A positive test for SARS CoV2 (with less than 25 Amplification Cycles) combined with a sick patient who displays the symptoms of acute Viremia and a CT Scan that shows Ground Glass Opacities (especially bilaterally) plus haematological findings of acute viral attack can then be part of the evidence for a preliminary (provisional) diagnosis of Covid 19. If the clinical course of the illness progresses as one would expect, then that diagnosis can become firmer over time.

This is how clinical medicine is practiced. The “epidemiologists” and public servant “medical advisers” to our governments are almost ALL non-clinicians. They never see a sick patient. They never take responsibility for treatment of a single person who is dangerously sick with Covid 19. They may be shocked to learn that clinicians generally ignore them and treat their sick patients with zinc, steroids (both inhaled, orally and IV), Vitamin D, Ivermectin and Hydroxychloroquine before the PCR test result comes back.

Molecular diagnostics are revolutionising the clinical practice of infectious disease. Their effects can be significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. Acute Care settings are NOT general population screening of the non-symptomatic. And PCR is not the only test or observation a clinician will use in an acute care setting.

With the evolution of novel molecular biology diagnostics tools (PCR), difficult questions have arisen regarding the role of such testing in the assessment of clinical infectious diseases. As molecular diagnostics continue to flow from bench to bedside , clinicians must acquire a working knowledge of the principles, diagnostic value, and limitations of varied assays in hospital-based settings.

The method relies on knowing at least partial sequences of the target DNA a priori and using them to design oligonucleotide primers that hybridise specifically to the target sequence. A partial sequence is not a complete sequence this is a potential problem for interpretation of the test results.

Through multiple cycles of heating and cooling in a thermocycler to produce rounds of target DNA denaturation, primer hybridisation, and primer extension, the target DNA is amplified exponentially“.

Theoretically, this method has the potential to generate billions of copies of target DNA from a single copy in less than one hour. Thus, a tiny fragment of dead (non-viable) genetic material can be found by PCR amplification methodology. You therefore have to be very careful about the results. They do not represent “cases” except on the BBC, the ABC and on CNN et al.

Limitations of PCR:

The principal shortcomings in applying PCR assays to the clinical setting include:

  • False positive results from background DNA contamination
  • The potential for false-negative test results
  • Detection sensitivity exceeding clinical significance
  • Limited detection space of the assay or platform for simultaneous identification of multiple species, virulence factors or drug resistance.

False positives:

The widespread use of PCR in clinical settings can be hampered largely by background contamination from exogenous sources of DNA. In most pathogen-specific assays, the predominant source of contamination is derived from “carryover” products from earlier PCR reactions which can be harboured and transmitted through PCR reagents, tubes, pipettes, and laboratory surfaces. Coupled with the robust amplification power of PCR, even very minor amounts of carry-over contamination may serve as substrates for amplification and lead to false-positive results.

Meticulous control measures such as good laboratory practices and physical separation of pre-amplification and post-amplification areas can reduce contamination risks but are not fool-proof. The use of enzymatic inactivation of carry-over DNA (i.e., uracil N-glycosylase) can further reduce contamination risk.

For front-line acute care physicians, or physicians working in disaster settings, a quick universal PCR assay, or panels of PCR assays targeting categories of pathogens involved in specific syndromes such as meningitis, pneumonia, or sepsis, can allow for rapid triage and early aggressive targeted therapy.

Because of rightful concern regarding disease transmission from asymptomatic and pre-symptomatic cases, this advice is not being followed. As a result, the great abundance of testing is screening not diagnostic.

One way to reduce false positive results is to repeat the test using a test with a different format (different manufacturer). Due to limited testing facilities, confirmation is not routinely performed and only a few positives are confirmed by a second rRT-PCR assay. It is likely that at current active disease prevalence the positive rRT-PCR results of many asymptomatic persons are false positives. We already know that the number is 90% at the very least and may be as high as 99%, especially if the Ct number exceeds 40.


if the two are equal or very nearly equal.)

The number of free primer molecules in Sample A after cycle 4.

The number of free primer molecules in Sample A after cycle 10.

The number of free primer molecules in Sample A after cycle 10.

The number of free primer molecules in Sample B after cycle 10.

The number of free TaqMan probe molecules in Sample A after cycle 10.

The number of free TaqMan probe molecules in Sample B after cycle 10.

The number of free TaqMan probe molecules in Sample A after cycle 18.

The r number of free TaqMan probe molecules in Sample B after cycle 18.

The number of reporter dye/mononucleotide complexes in Sample A after cycle 18.

The number of reporter dye/mononucleotide complexes in Sample B after cycle 18.

The number of amplified HER2 fragments in Sample A after cycle 20.

The number of amplified HER2 fragments in Sample B after cycle 20.

The number of amplified HER2 fragments in Sample A after cycle 30.

The number of amplified HER2 fragments in Sample B after cycle 30.


About those RT-PCR Tests

Reiner Fuellmich is about to instigate legal proceedings against the perpetrators of the Covid-19 scamdemic: covid-fraud-lawyers-medical-experts-start -legal-proceedings-against WHO His case rests principally on the inappropriate use of the RT-PCR test. A colleague of mine has graciously agreed for me to reproduce his assessments of this test – he is not alone. Hat Tip Gerry @ http://boomfinanceandeconomics.com/#/

PCR is a difficult technology to grasp because there are so many subtle aspects. PCR technology has been around since 1987 when Kary Mullis invented it. He won the Nobel Prize for doing so. It is not a test that clinicians order lightly. In normal clinical medical practice, it is ordered only when dealing with very ill patients. Careful consideration and very careful interpretation are needed and the clinical situation is critical.

Everyone should watch the videos of Kary Mullis on YouTube and Bitchute. He was a genius and brilliant scientist unfortunately, he died recently — just before the Covid outbreak . https://www.bitchute.com/video/8KsH34IGgqBw/

Quote from Kary Mullis, Nobel Prize winner and inventor of PCR Tests: “Guys like Fauci get up there and start talking, you know, he doesn’t know anything really about anything and I’d say that to his face. Nothing. The man thinks you can take a blood sample and stick it in an electron microscope and if it’s got a virus in there you’ll know it. He doesn’t understand electron microscopy and he doesn’t understand medicine and he should not be in a position like he’s in. Most of those guys up there on the top are just total administrative people and they don’t know anything about what’s going on in the body. You know, those guys have got an agenda, which is not what we would like them to have being that we pay for them to take care of our health in some way. They’ve got a personal kind of agenda. They make up their own rules as they go. They change them when they want to. And they smugly, like Tony Fauci does not mind going on television in front of the people who pay his salary and lie directly into the camera.”

The number of PCR cycles can have dramatic consequence. This is the kernel of the issue although there are many, many more factors. PCR tests cannot distinguish between live (viable) micro-organisms and dead ones. They both contain genetic material. This is why using PCR tests with high Ct numbers (Amplification Cycle Numbers) in a Screening situation is ridiculous. It must never be used in non-symptomatic people to “diagnose” illness.

Because of this problem, it can never be regarded as a purely “diagnostic test”. No clinicians worth their salt will ever regard a stand-alone PCR test as diagnostic. We have over 20 years of clinical experience with this technology. If you do more than 30 Amplification Cycles, you are guaranteed to find non-viable (dead) viral remnants or contaminants. Many nations have been doing 40 – 45 cycles during the Covid phenomenon. The cycle number is guaranteed to produce a high rate of positive tests that are, in fact, false. We call them “False Positives” in the world of clinical medicine.

A positive test for SARS CoV2 (with less than 25 Amplification Cycles) combined with a sick patient who displays the symptoms of acute Viremia and a CT Scan that shows Ground Glass Opacities (especially bilaterally) plus haematological findings of acute viral attack can then be part of the evidence for a preliminary (provisional) diagnosis of Covid 19. If the clinical course of the illness progresses as one would expect, then that diagnosis can become firmer over time.

This is how clinical medicine is practiced. The “epidemiologists” and public servant “medical advisers” to our governments are almost ALL non-clinicians. They never see a sick patient. They never take responsibility for treatment of a single person who is dangerously sick with Covid 19. They may be shocked to learn that clinicians generally ignore them and treat their sick patients with zinc, steroids (both inhaled, orally and IV), Vitamin D, Ivermectin and Hydroxychloroquine before the PCR test result comes back.

Molecular diagnostics are revolutionising the clinical practice of infectious disease. Their effects can be significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. Acute Care settings are NOT general population screening of the non-symptomatic. And PCR is not the only test or observation a clinician will use in an acute care setting.

With the evolution of novel molecular biology diagnostics tools (PCR), difficult questions have arisen regarding the role of such testing in the assessment of clinical infectious diseases. As molecular diagnostics continue to flow from bench to bedside, clinicians must acquire a working knowledge of the principles, diagnostic value, and limitations of varied assays in hospital-based settings .

The method relies on knowing at least partial sequences of the target DNA a priori and using them to design oligonucleotide primers that hybridise specifically to the target sequence. A partial sequence is not a complete sequence this is a potential problem for interpretation of the test results.

Through multiple cycles of heating and cooling in a thermocycler to produce rounds of target DNA denaturation, primer hybridisation, and primer extension, the target DNA is amplified exponentially“.

Theoretically, this method has the potential to generate billions of copies of target DNA from a single copy in less than one hour. Thus, a tiny fragment of dead (non-viable) genetic material can be found by PCR amplification methodology. You therefore have to be very careful about the results. They do not represent “cases” except on the BBC, the ABC and on CNN et al.

Limitations of PCR: The principal shortcomings in applying PCR assays to the clinical setting include:

  • False positive results from background DNA contamination
  • The potential for false-negative test results
  • Detection sensitivity exceeding clinical significance
  • Limited detection space of the assay or platform for simultaneous identification of multiple species, virulence factors or drug resistance.

False positives: The widespread use of PCR in clinical settings can be hampered largely by background contamination from exogenous sources of DNA. In most pathogen-specific assays, the predominant source of contamination is derived from “carryover” products from earlier PCR reactions which can be harboured and transmitted through PCR reagents, tubes, pipettes, and laboratory surfaces. Coupled with the robust amplification power of PCR, even very minor amounts of carry-over contamination may serve as substrates for amplification and lead to false-positive results.


Why is the number of PCR cycles limited? - Biology

The Polymerase Chain Reaction ( PCR )

PCR begins with a mixture containing a dsDNA template , a pair of short ssDNA oligonucleotide primers , a pool of the four dNTPs , and a heat-resistant DNA polymerase, Taq Enzyme . The reaction is carried out in a computer-regulated heating block, a thermal cycler , which permits rapid, controlled heating & cooling. The primers are chosen so that they are base-complementary to opposite ends of either strand of a short stretch of DNA containing the gene region of interest: PCR thus requires some prior knowledge of the gene.

The reaction is first heated to 95 o C to melt (denature) the dsDNA into separate strands. The reaction is then cooled to

50 o C, at which temperature the primers will find base-complementary regions in the ssDNA, to which they will stick (anneal). The reaction is finally heated to 72 o C, at which temperature the Taq enzyme replicates the primed ssDNA (extension). At the end of one cycle, the region between the two primers has been copied once, producing two copies of the original gene region. [This is slightly oversimplified: see details].

Because a heat-resistant polymerase is used, the reaction can be repeated continuously without addition of more enzyme. Each cycle doubles the copy number of the amplified gene: ten cycles ideally produces 2 4 8 16 32 64 128 256 512 1,024 ( 2 10 ) copies. Thus, 30 cycles yields a ( 2 10x3 ) = 10 9 -fold amplification. This produces a sufficient quantity of the gene region of interest for direct analysis, for example by DNA sequencing.


DNA binding dye:

For a novice and inexperienced person, the DNA binding dye method is the best technique for real-time detection.

The dye has its own fluorescence. Once the dye bind to the double-stranded DNA the fluorescence emitted by the dye increases 100 to 1000 fold than the original signal.

However, the original dye fluorescence is taken as the baseline for the detection.

The method is rapid, quick, reliable and cost-effective. Also, the chance of error in the experiments are less and the reaction set up is simple and easy to use.

The result of the experiment depends on the specificity of the primers used in the PCR reaction.

Because even though, the primers are bind non-specifically, the DNA binding dye binds to the non-specific sequence and gives the fluorescent signals.

Because the dye detects the double-stranded DNA to binds, even if the dsDNA is non-specific, the dye must bind to it.

Therefore the chance of the non-specific detection is high in the SYBR green dye-based method.

The SYBR green is one of the most popular dyes used in real-time PCR.

The sensitivity of the experiment is limited. Again a question arise in mind,

Is it suitable for the determination of sensitive templates?

We can identify the non-specific bindings into the reaction by doing the melting curve analysis.

Melting curve analysis:

Once the amplification reaction is completed and the fluorescence signals are recorded, the template is melted for the determination of the non-specific bindings.

The template is melted using heating, the dye dissociates and the fluorescence signals are reduced.

The decreased transition of a wide range fluorescence is reported for the specific product while different heat transition recorded for different short non-specific bands.

“Larger sequences takes more time and higher temperature for melting while non-specific bands melt at a lower temperature and has different melting temperature curves.”

The data are plotted in fluorescence vs melting temperature graph below.

The fluorescence vs melting temperature graph is also called a dissociation curve and the method is called a dissociation curve analysis.

Image: The image shows the dissociation curve for specific product and primer dimers while another image shows different dissociation curves for two homozygous and a heterozygous.

SYBR green and EvaGreen are two main dye used in the quantitative real-time PCR. The experiments are used in the validation of the assays such as DNA microarray.

The TaqMan probe chemistry is widely used in the quantitative PCR, the TaqMan name is taken from the “Taq” Taq DNA polymerase (because the probe chemistry depends on the activity of Taq DNA polymerase) and “Man” from the PacMan game. Yes, the name is taken from the game. Remember what PacMan do?


Different types of PCR technique based on thermocycling (heating and cooling steps)

Thermocycling techniques use temperature cycling to drive repeated cycles of DNA synthesis.

Multiplex PCR

Multiplex PCR is a type of PCR technique which allows an amplification of many target sequences concurrently in the same reaction mixture.

A single reaction mixture includes sets of primer pairs for different DNA targets. It reduces the consumption of PCR reagents, and, at the same time, imposes restrictions on used primers.

To work properly within one reaction, sets of primers must be optimized. They have to have similar annealing temperatures and produce amplicons of different sizes to form distinct gel electrophoresis bands for the followed PCR analysis.

Nested PCR

Nested PCR is used to increase the specificity of a DNA amplification reducing unspecific products.

This technique utilizes two sets of primers.

The first set allows a first polymerase chain reaction. The product of this reaction serves as a source of target DNA to a second PCR using the second set of primers.

Hot-start PCR

This type of polymerase chain reaction serves to reduce non-specific amplification during the initial set up stages.

Hot-start PCR technique keeps the DNA polymerase in an inactive state at temperatures lower than an annealing temperature.

This modification prevents the amplification during reaction setup when primers bind to DNA sequences with low homology.

Two variants of this technique are mechanical and non-mechanical hot start PCR.

Mechanical hot start PCR performed by heating the reaction mixture to the DNA melting temperature before adding the Taq polymerase.

Non-mechanical hot start PCR uses specialized enzyme systems which inhibit an activation of the DNA polymerase at room temperature.

Touchdown PCR

Touchdown PCR is another technique to reduce nonspecific amplification.

It is achieved by raising the annealing temperature above the melting temperature of the used primers in the initial cycles and lowering in the later cycles.

The higher temperatures during the initial cycles help primers to bind to DNA templates with greater specificity while the lower temperatures allow more efficient amplification from the produced amplicons.

Ligase Chain Reaction (LCR)

This type of PCR technique uses four primers for DNA amplification (two primers for each strand of the DNA target).

Ligase Chain Reaction primers are much longer than usual PCR primers and designed to cover the entire sequence to be amplified.

During the first annealing step, primers are sealed by a thermostable DNA ligase.

This generates a fragment that is as long as the total length of each pair of primers which serves as the DNA templates for subsequent cycles.

The main advantage of Ligase Chain Reaction is that a single point mutation near the junction in the original template DNA can prevent the reaction and an absence of product can be an indicator of mutations.

Quantitative PCR (qPCR)

The amount of product that is synthesized during a set number of cycles of a polymerase chain reaction depends on the number of DNA molecules that are present in the starting mixture. This enables PCR to be used to quantify the number of DNA molecules present in an extract.

In quantitative PCR the amount of product synthesized during a test PCR is compared with the amounts synthesized during PCRs with known quantities of starting DNA.

Real-time PCR

Today, quantification is carried out by real-time PCR - a modification of the standard PCR technique in which synthesis of the product is measured over time.

More frequently this method is used to measure RNA amounts.

For example to determine the expression of a particular gene in cancerous cells. This method allows monitoring the development of cancer.

Reverse transcription PCR

To carry out polymerase chain reaction where RNA is the starting material this method uses reverse transcriptase, a process called RT–PCR (reverse transcriptase polymerase chain reaction).

The first step in this method is to convert the RNA molecules into single-stranded complementary DNA (cDNA). After this step, the experiment proceeds as in the standard technique.

Some thermostable polymerases, such as Tth, have a reverse transcriptase activity under certain buffer conditions and able to make DNA copies of both RNA and DNA molecules.

TaqMan PCR

TaqMan PCR is one of the real-time PCR techniques.

It uses an oligonucleotide probe which is complementary to an internal sequence within the amplified strands.

It has a fluorescent group at one end and a quencher at another end. As long as both fluorophore and quencher stay within the oligonucleotide probe, no fluorescence is emitted.

During DNA amplification, the oligonucleotide probe, and the primers will bind to newly synthesized strands. The polymerase will destroy the probe due to the intrinsic 5′→3′ exonuclease activity and release the fluorophore.

The intensity of the fluorescence is proportional to the amount of generated product.

Assembly PCR

Assembly PCR or Polymerase Cycling Assembly was developed to produce novel long nucleic acid sequences.

The main difference from traditional polymerase chain reaction is the length and quantity of primers.

To synthesize artificial oligonucleotide, assembly PCR is performed on long, up to 50 nucleotides, primers. These primers have short overlapping segments and alternate between sense and antisense directions covering the entire target sequence.

During successive cycles, the primers hybridize by complementary segments and then polymerase increases the length of fragments producing the final long nucleic acid sequence.

Typically, the assembly phase is followed by a regular polymerase chain reaction with end primers to increase the amount of the final product.


Troubleshooting your PCR

What should you do when your PCR goes wrong? The FAQs below can set you on a path toward successful PCR.

For tips on how to rescue your experiments from PCR contamination, check out this blog article.

If no amplification products are obtained, what parameters should be considered first when troubleshooting?

Consider the following:

  • First, ensure that all PCR components were included in the reactions. A positive control should always be included to ensure that each component is present and functional.
  • If there were no problems with the experimental setup, increase the number of PCR cycles (3&ndash5 cycles at a time), up to 40 cycles. Increasing the cycle number can overcome issues with a low-abundance template or template inaccessibility due to impurities in or poor priming efficiency of the primers.
  • If increasing the cycle number does not improve results, the PCR conditions might be too stringent for the particular primer set or template. Consider modifying the PCR conditions as follows:
    • Lower the annealing temperature in increments of 2 degrees.
    • Increase the extension time.
    • Increase the template amount. Refer to the guidelines provided with the enzyme to determine the optimal amount of template.

    Consider these additional possible reasons for PCR failure:

    • PCR inhibitors in the template sample
      If PCR inhibitors are present, using diluted template may increase PCR efficiency. Alternatively, the template may need to be purified using a kit such as the NucleoSpin Gel and PCR Clean-up kit. If purifying the template is not a possibility, an enzyme that has a higher tolerance to impurities, such as Terra PCR Direct polymerase, may improve results.
    • The template has >65% GC content
      When amplifying from templates with high GC content, use an enzyme formulated for this condition. Visit our PCR selection guide to find an appropriate enzyme.
    • Primers are not optimal
      Check your primers carefully redesign if necessary. Also, consider re-amplifying the primary PCR product using 10-fold dilutions (1:100 to 1:10,000) using nested primers.

    When using PrimeSTAR HS DNA Polymerase, consider:

    • Using an appropriate amount of template. If the template is human genomic DNA or a cDNA library, use no more than

    When using PrimeSTAR Max DNA Polymerase, consider:

    • Adjusting the extension time if the reaction mixture contains excess template. If the amount of template exceeds 200 ng in a 50-µl reaction mixture, set the extension time between 30 sec/kb and 1 min/kb.
    • Increasing the concentration of the primers.

    When using SpeedSTAR HS DNA Polymerase, consider:

    • Increasing the extension time. Although the standard extension time is 10 sec/kb, the extension time can be increased to

    If there are nonspecific amplification bands, what can be done to improve specificity?

    All Takara Bio PCR polymerases

    Issue:

    Solution:

    Use BLAST alignment to determine if the 3' ends of the primers are complementary to sites other than the target site(s). Redesign primers if necessary or modify PCR conditions.

    Issue:

    PCR conditions are not sufficiently stringent.

    Solutions:

    • Increase the annealing temperature in increments of 2 degrees.
    • Use touchdown PCR.
    • Use a two-step PCR protocol.
    • Reduce the number of PCR cycles.

    Issue:

    Too much template was used.

    Solution:

    Reduce the amount by 2&ndash5 fold.

    PrimeSTAR HS and PrimeSTAR Max DNA polymerases

    Issue:

    Annealing time is too long.

    Solution:

    To achieve specific amplification, it is essential to use a short annealing time (5&ndash15 sec) when performing three-step PCR.

    PrimeSTAR GXL DNA polymerases

    Issue:

    Primers have suboptimal Tm values.

    Solution:

    To amplify targets <1 kb, design primers with Tm values >55°C, and use an annealing temperature of 60°C. If the primer Tm values are <55°C, try a shorter extension time between 5 and 10 sec/kb.

    Takara Ex Taq and Takara LA Taq DNA polymerases

    Issue:

    Nonspecific primer annealing at low temperatures.

    Solution:

    The hot-start versions of these enzymes may improve results for some primers.

    SpeedSTAR HS DNA Polymerase

    Issue:

    Smearing of the PCR product bands on a gel.

    Solution:

    Excessively long extension times may result in smearing. The general recommendation for extension time for this enzyme is 10&ndash20 sec/kb. If PCR yield is low, try increasing the number of cycles by 5.

    If PCR generates a smear after running the products on a gel, what can be done to improve the results?

    First, determine the source of the smear using positive and negative (no template) controls. This can determine if the cause of the smear is contamination or overcycling, or if the smear results from poorly designed primers or suboptimal PCR conditions.

    If the negative control is blank, there is no contamination. Instead, the PCR conditions will need to be optimized consider the following when adjusting the PCR conditions:

    • Reduce the amount of template.
    • Increase the annealing temperature.
    • Use touchdown PCR.
    • Reduce the number of PCR cycles.
    • Redesign the primers.
    • Use nested primers.
    • Re-amplify the product. (A small plug of the gel can be removed with a micropipette tip, and the DNA can be recovered by adding the plug to 200 µl of water and then incubating at 37°C. 5 µl of this solution can be used as PCR template for re-amplification.)

    If the negative control is also smeared, there is contamination. You will need to determine the source of this contamination. It may be necessary to replace PCR reagents and to decontaminate pipettes and your workstation (see questions below for more information on contamination).

    What are some sources of PCR contamination?

    There are four main sources of PCR contamination:

    1. The most common source of contamination is PCR product from previous amplifications (called "carryover contamination"). When large amounts of PCR product (10 12 molecules) are generated repeatedly over a period of time, the potential for contamination increases.
    2. Another source of contamination is cloned DNA previously handled in the laboratory.
    3. Sample-to-sample contamination can also occur. This source of contamination is most likely to be found in samples that require extensive processing prior to amplification.
    4. Reactions can also be contaminated with exogenous DNA in the environment, including DNA present on laboratory equipment and in reagents used for DNA extraction and PCR.

    How can contamination be avoided?

    The sensitivity of PCR requires that samples are not contaminated with any exogenous DNA or any previously amplified products from the laboratory environment. We recommend that distinct areas are used for sample preparation, PCR setup, and post-PCR analysis.

    A laminar flow cabinet equipped with a UV lamp is recommended for preparing reaction mixtures. Two stations should be established that are physically separated from each other.

    • Establish a "pre-PCR area" that is for PCR reaction setup only. No items from the "post-PCR area" should be introduced into this area this includes items such as notebooks and pens.
    • Establish a "post-PCR area" that is used for PCR, purifying PCR-amplified DNA, measuring DNA concentration, running agarose gels, and analyzing PCR products.

    Equipment should also be restricted to these areas. The PCR machine and electrophoresis apparatus should be located in the post-PCR area. Having pipettes and pipette tips with aerosol filters dedicated for DNA sample and reaction mixture preparation only is strongly recommended. Additional recommendations include:

    • Having separate sets of pipettes and pipette tips, lab coats, glove boxes, and waste baskets for the pre-PCR and post-PCR areas.
    • Labeling pre- and post-PCR items, so they are not removed from their designated work area.
    • Following the golden rule of PCR: NEVER bring any reagents, equipment, or pipettes used in a post-PCR area back to the pre-PCR area.
    • Preparing and storing reagents for PCR separately and using them solely for their designated purpose. Reagents should be aliquoted in small portions and stored in designated areas depending on if they are used for pre-PCR or post-PCR applications. The aliquots should be stored separately from other DNA samples.

    A control reaction that omits template DNA should always be performed to confirm the absence of contamination. In addition, the number of PCR cycles should be kept to a minimum, as highly sensitive assays are more prone to the effects of contamination.

    How can I decontaminate if I have PCR contamination?

    1. Leave pipettes under UV light in the cell culture hood overnight. UV irradiation promotes cross-linking of thymidine residues, damaging residual DNA.
    2. Spray workstations/equipment/pipettes with 10% bleach and then wipe clean.
    3. Change workstations move the pre-PCR area to another pre-cleaned location.
    4. Do not use any instruments or pipettes you have used before.

    What can I do if the PCR generates errors?

    To avoid errors during PCR, we recommend using a high-fidelity enzyme (see selection guide). In addition, be sure to avoid the following:

    1. Overcycling
      Overcycling PCR reactions often:
      • Changes the pH of the reaction in a manner that destabilizes DNA.
      • Increases the amount of PCR product, thereby reducing the efficiency of the polymerase and promoting errors.
      • Decreases the amount of dNTPs, thereby increasing the likelihood of base misincorporation due to the unbalanced dNTP concentration. (If using Takara Bio's PCR enzymes, the dNTP concentration is optimized to 200 nM increasing dNTP concentration leads to misincorporation.)
      • Causes accumulation of single-stranded and double-stranded DNA.
    2. High Mg 2+ concentration
      Mg 2+ concentration ranges from 1&ndash5 mM. Using a high Mg 2+ concentration may increase yield, but it might also impact the proofreading activity of enzymes. However, the Mg 2+ concentration should always be higher than the dNTP concentration.
    3. Template DNA damage
      Limit UV exposure time when analyzing or excising PCR products from gels.

    What are PCR inhibitors?

    Impurities that interfere with PCR amplification are known as PCR inhibitors. PCR inhibitors are present in a large variety of sample types and may lead to decreased PCR sensitivity or even false-negative PCR results. PCR inhibitors may have both inorganic and organic origins (Schrader 2012).

    Inorganic PCR inhibitors include:

    • Calcium or other metal ions that compete with magnesium
    • EDTA that binds to magnesium, reducing its concentration

    Some organic PCR inhibitors include:

    • Polysaccharides and glycolipids that mimic the structure of nucleic acids, interfering with primers binding to the template
    • Melanin and collagen that form a reversible complex with DNA polymerase
    • Humic acids that interact with template DNA and polymerase, preventing the enzymatic reaction, even at low concentrations
    • Urea that may lead to degradation of the polymerase

    Other organic compounds that can inhibit PCR include:

    • Hemoglobin, lactoferrin, and IgG in blood, serum, or plasma samples
    • Anticoagulants such as heparin
    • Polyphenols, pectin, and xylane from plants
    • Ethanol, isopropyl alcohol, phenol, or detergents such as SDS

    If inhibitors are present in the template preparation, a 100-fold dilution of the starting template may sufficiently dilute the inhibitor and allow amplification. Alternatively, ethanol precipitation of the template may be needed to resolve the problem.

    References

    Schrader, C., et al. PCR inhibitors&mdashoccurrence, properties and removal. J Appl Microbiol. 113:1014&ndash1026 (2012).

    What is PCR overcycling? How do I know if my product is overcycled?

    PCR overcycling is when cycling goes beyond the exponential phase of amplification. Overcycling occurs when the following events take place during PCR:

    • Depletion of substrates (dNTPs or primers).
    • The reagents (dNTPs or enzymes) are no longer stable at the denaturation temperature.
    • The PCR polymerase is inhibited by the product (pyrophosphate, duplex DNA).
    • Competition for reagents (dNTPs and primers) by nonspecific products.
    • Lowering of the pH of the reaction.
    • Incomplete denaturation/strand separation of products at high product concentrations.

    The indicator of PCR overcycling is an intense background smear with indistinguishable bands when the reaction is resolved on an agarose gel. It is always recommended to perform a preliminary test to determine the minimal number of PCR cycles needed to yield a sufficient product. The PCR product remains in the linear phase of amplification if the product yield is noticeably increased every 3&ndash5 cycles. We find that overcycled cDNA does not produce suitable template for any downstream application.

    What types of mutations can be caused by PCR?

    PCR polymerases can introduce different types of mutations, including single-base substitutions, deletions, and insertions. Base substitutions are typically caused by misincorporation of an incorrect dNTP during DNA synthesis.

    Polymerases may generate mutations at locations where one or more nucleotides are lost or gained. The frequency of this type of mutation can be sequence dependent, and might be higher in highly repetitive sequences. The most common mutation is a loss of a single nucleotide, which could be a result of template-primer misalignment within a repetitive homopolymeric sequence. DNA rearrangements can also occur when the polymerase terminates synthesis on one DNA strand and continues synthesis after priming occurs on a complementary strand (i.e., strand-switching or jumping PCR). This type of mutation takes place when there is high homology between different regions of DNA. Excessive DNA template in the reaction may also promote this type of mutation.

    What factors contribute to PCR-introduced mutations?

    The following factors can contribute to PCR-introduced mutations:

    • Unbalanced dNTP concentrations
      Unequal amounts of the four dNTPs can increase base substitution by as high as eight-fold. Using equal concentrations of the four dNTPs is critical for reducing the error rate of the polymerase.
    • High enzyme concentration
    • Long incubation times
    • Lack of 3'&rarr5' exonuclease activity
    • Magnesium concentration
      Fidelity is highest when the concentration of Mg 2+ is equimolar to the total concentration of the dNTPs. Fidelity decreases when the concentration of free divalent cations increases.
    • pH of the reaction
      Lowering the pH of the reaction by three units can increase base substitutions up to 60-fold. Low pH (<6.0) may lead to spontaneous purine loss.
    • DNA damage
      DNA damage can occur at high temperatures, possibly increasing the rate of mutation. One frequent mutation is deamination of cytosine to produce uracil.
    • The presence of A stretches in primer sequences
    • Overcycling
      Error rate is increased when the DNA concentration is increased during the final PCR cycles. The total number of cycles should be kept to a minimum to produce the desired PCR product without errors.

    What are PCR artifacts?

    The following are common PCR artifacts:

    • Primer dimers
      Primer dimers are formed through self-complementarity at the 3' end of the amplification primers. Primer dimers are suspected if product is produced in a template-free reaction (negative control). To avoid primer dimers, primers shouldn't have complementarity at their 3' ends.
    • Chimeric PCR products
      Chimeric PCR products can be caused by incompletely extended template. In other words, single-stranded template that was not completely replicated due to premature polymerase termination can anneal to partially homologous template. This creates chimeric PCR products. To minimize chimeras, use the fewest possible PCR amplification cycles.
    • PCR bias
      PCR bias occurs when some sequences are amplified more efficiently than others due to preferential binding by PCR primers. If one sequence is amplified 10% more than another in one cycle, it will be 17.4-times more abundant after 30 cycles. To reduce PCR bias, use a high ramp rate between the denaturation and annealing steps and use low annealing temperatures. Long extension times (>180 sec) should be avoided.
    • PCR drift
      PCR drift is due to stochastic fluctuation in the interactions of PCR reagents, particularly in the early cycles when a very low template concentration exists. This artifact is observed in multiplex assays, where a loss of sensitivity is caused by the interactions between different sets of primers. It is important to carefully design primers for these types of assays.
    • PCR generates high-molecular-weight products that barely migrate through the agarose gel
      There is no good explanation for this artifact. Most researchers assume that this is caused by overcycling, since in the later stages of PCR, both single- and double-stranded molecules accumulate. Accumulation of such single-stranded molecules can create heteroduplexes by competing with the primers. Incomplete denaturation in later stages, when there is a high concentration of PCR products, prevents DNA strand separation, and thus a newly formed amplicon may remain bound to the previously made template. This process could repeat, trapping PCR products in a network of molecules.

    Another explanation for the origin of high-molecular-weight smears is the partial extension of templates during initial PCR cycles. Partial extensions could be generated by jumping artifacts&mdashwhen a primer or single-stranded DNA anneals and extends from one priming site, then anneals partially to a homologous segment elsewhere (see Chimeric PCR products, above). Partially extended molecules can act as new primers, since they contain a free 3'-OH, and could generate chimeric molecules that combine the initial priming site and the "jump" site.

    Finally, this type of artifact can also be generated when a crude template is used for PCR. Products amplified directly from animal or plant tissues can become trapped in cell debris, which prevents them from migrating in the gel. This problem can be solved by Proteinase K digestion of the amplified PCR product:

    • Before loading your samples onto a gel, add 1 µl of the loading buffer containing Proteinase K to 4 µl of the PCR reaction.

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    Polymerase Chain Reaction (PCR) | Biotechnology

    The below mentioned article provides a Beginner’s Guide to Polymerase Chain Reaction (PCR). After reading this article you will learn about: 1. History of the PCR 2. The Principle of Polymerase Chain Reaction 3. Requirements of PCR 4. The PCR Reaction Cycle 5. Analysis of PCR Products 6. Applications 7. Precautions and Drawbacks 8. Modifications.

    1. History of the PCR
    2. The Principle of Polymerase Chain Reaction
    3. Requirements of PCR
    4. The PCR Reaction Cycle
    5. Analysis of PCR Products
    6. Applications of PCR
    7. Precautions and Drawbacks
    8. Modifications

    1. History of the PCR:

    The idea for PCR is credited to Kary-Mullis who was a research scientist in 1980s at a Cali­fornia biotechnology company called Cetus. Mullis, and five other researchers in Human Genetics Department at Cetus, demonstrated that oligonucleotide primers could be used to specifically amplify defined segments of ge­nomic DNA (or cDNA). Mullis was co-winner of 1993 Nobel Prize in Chemistry.

    2. The Principle of Polymerase Chain Reaction:

    Polymerase chain reaction (PCR) is a primer mediated enzymatic amplification of specifi­cally cloned or genomic DNA sequences. In this process we take the DNA with a target se­quence which we want to amplify, denature it by increasing the temperature and then use a sequence specific primer for the amplification of our target sequence by the help of a ther­mos-table DNA polymerase.

    In this technique we try to reproduce an artificial environment under in vitro conditions in which the target DNA sequence undergoes multiple rounds of replication cycle to produce an enormous cop­ies of our target gene.

    The process PCR has three fundamental steps:

    Step 1:

    Double-stranded DNA template denaturation by increasing the tempera­ture to 94 – 98°C for 30 seconds for 2 mi­nutes.

    Step 2:

    Annealing of two oligonucleotide primers to the single-stranded tem­plate by lowering the temperature to 50 – 65°C.

    Step 3:

    Enzymatic extension of primers to produce copies that can serve as tem­plates in subsequent cycles.

    3. Requirements of PCR:

    (a) DNA Template:

    The original DNA mol­ecule that is to be copied is called the DNA template and the segment of it that will actually be amplified is known as the tar­get sequence. A trace amount of the DNA template is sufficient. This can be obtained by any one of the DNA isolation techniques discussed before.

    (b) PCR Primers:

    Two PCR primers are needed to initiate DNA synthesis. These are short pieces of single-stranded DNA that match the sequences at either end of the target DNA segment. PCR primers are made by chemical synthesis of DNA.

    There are several computer programs available to suggest suitable primers for the process of PCR, and some of the general guide­lines are listed below:

    Shorter primers have a tendency to go and anneal to the non-target sequence of the DNA template. This will result in production of DNA copies of having non-target sequence. The greater the complexity of the template DNA, the more likely this is to happen.

    Thus, a Short primer may offer sufficient specificity when amplifying using a simple template such as a small plasmid, but a long primer may be re­quired when using eukaryotic genomic DNA as template. In practice, 20-30 nucleotides is generally satisfactory.

    Primers do not need to match the template completely, although the 3′ end of the primer should be correctly base-paired to the template, otherwise the polymerase will not be able to extend it. It is often beneficial to have C or G as the 3′ terminal nucleotide. This makes the binding of the 3′ end of the primer to the tem­plate more stable than it would be with A or T at the 3′ end.

    3. Melting Temperature:

    The temperatures at which the two primers can associate with the template should be rela­tively similar to ensure that they both bind at about the same time as temperatures are be­ing lowered during annealing. The similarity of melting temperatures is likely to mean that the primers have a similar nucleotide compo­sition.

    4. Internal Secondary Structure:

    This should be avoided in order to prevent the primer to fold back on itself and not be avail­able to bind to the template.

    5. Primer-Primer Annealing:

    It is also important to avoid the two primers being able to anneal to each other. Extension by DNA polymerase of two self-annealed prim­ers leads to formation of a primer dimer.

    (c) Thermo-Stable DNA Polymerase:

    The enzyme DNA polymerase is needed to manufacture the DNA copies. The Klenow fragment was the first DNA polymerase enzyme used in PCR. The Klenow frag­ment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin.

    After enzymatic modification it retains the 5′-3′ polymerase activity and the 3′ → 5′ exonuclease activity for re­moval of pre-coding nucleotides and proof­reading, but loses its 5′ → 3′ exonuclease activity.

    Klenow fragment failed to play a successful role as a polymerase enzyme for lacking a stability at high temperature. As we know that the PCR procedure involves several temperature steps, in this situa­tion we had to replenish the Klenow frag­ment during each cycle.

    To solve this is­sued heat resistant DNA polymerase was required. This came originally from heat resistant bacteria living in hot springs at temperatures up to 90°C. Today Taq poly­merase from Thermusaquaticus is the most widely used PCR DNA polymerase enzyme. It is generally produced by expres­sion of the gene in E. coli.

    The thermo-sta­bility of the Taq enzyme helps in its puri­fication after expression in E. coli, since- contaminating E. coli proteins can be in­activated by heating. The enzyme has 5′- 3′ DNA polymerase and 5′-3′ exonuclease activities. It will polymerize about 50-60 nucleotides per second. However, the en­zyme has a number of properties that may be disadvantageous.

    1. Taq Polymerase has No Proof-Reading (3′-5′ exonuclease) Activity:

    Consequently about one nucleotide in 10 4 in­corporated is incorrect and the individual prod­ucts of PCR will be a heterogeneous popula­tion.

    2. Taq Polymerase has Relatively Low Processivity:

    This means that it is likely to dissociate from the template before it has synthesized a long piece of DNA.

    3. Taq Polymerase is not Fully Heat Stable:

    It has a half-life of about 40 min at 95°C, which means there will be significant loss of activity over the 30 or so cycles used in a typical PCR experiment. It may, therefore, be necessary to add more enzyme during the course of an ex­periment.

    4. Taq Polymerase Incorporates an Ex­tra a Residue:

    This is incorporated on the 3′ end of the mole­cule synthesized, and is not template encoded. A number of polymerases are available from other Thermus species. These include Tfl and Tth enzymes from Thermusflavus and Thermusthermophilus respectively.

    These generally do not have 3′-5′ proof-reading ac­tivity. Polymerases are also available from other genera of bacteria (including archaebacteria), and many of these enzymes have 3′-5′ proof-reading activity (which also means, they do not usually add terminal nucleotides that are not template directed).

    Proof-reading en­zymes include Tli from Thermococcuslitoraiis and Pfu from Pyrococcusfuriosus. These marine bacteria generally grow at even higher temperatures than Thermusaquaticus, and the polymerases are more ther­mo-stable than the Taq enzyme.

    (d) Deoxy Nucleotide Triphosphates:

    A supply of four deoxynucleotide triphos­phates, dATP, dCTP, dGTP and dTTP, are needed by the polymerase to make the new DNA.

    (e) PCR Machine:

    Finally we need a PCR machine to keep changing the tempera­ture. The PCR process requires cycling through sev­eral different temperatures. Because of this, PCR machines are sometimes called thermo-cyclers.

    4. The PCR Reaction Cycle:

    The PCR is a chain reaction because newly synthesized DNA strands will acts as template for further DNA synthesis in subsequent cycle. After 25 cycles of DNA synthesis, the products of the PCR will include, in addition to the start­ing DNA, about 10 5 copies of the specific tar­get sequence.

    PCR consists of a series of cycles of three successive reactions:

    Denaturation Step:

    This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes the melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

    Annealing Step:

    The reaction tempera­ture is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single- stranded DNA template. Typically the anneal­ing temperature is about 3-5 degrees Celsius below the Tm of the primers used.

    The Tm can be determined experimentally or calculate from the following formula:

    Tm = (4 x [G + C]) + (2 x [A + T])°C

    Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The poly­merase binds to the primer-template hybrid and begins DNA synthesis.

    Extension/Elongation Step:

    The tem­perature at this step depends on the DNA poly­merase used. Taq polymerase has its opti­mum activity temperature at 75-80°C, and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5′ to 3′ direction.

    The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute.

    Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.

    For example, if one starts with a single double-stranded DNA molecule, after 20 cycles the number of mol­ecules synthesized by PCR becomes 1吆 6 , and after 30 cycles the number of the DNA mol­ecules increases to 1吆 9 .

    This number can be calculated by the help of following formula:

    where Mf is the final number of DNA mol­ecules produced by PCR, Mf is the initial amount of DNA molecules, and n is the num­ber of PCR cycles.

    Final Elongation:

    This single step is oc­casionally performed at a temperature of 70- 74 °C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

    5. Analysis of PCR Products:

    PCR is often used as a technique to gain infor­mation about the DNA template carrying a specific target sequence. Biotechnology re­search widely depends upon PCR at various situations. Hence there are several methods for analysing the products of PCR.

    Following three techniques are important:

    (a) Gel Electrophoresis of PCR Products:

    The final results of most PCR experiments are confirmed by subjecting a portion of the amplified reaction mixture to agarose gel electrophoresis. This can inform us the validity of the PCR experiment. If the ex­pected band during gel visualization is absent, or if additional bands are present, something has gone wrong and the experi­ment must be repeated.

    In some cases, agarose gel electrophoresis is used not only to determine whether a PCR experiment has worked, but also to obtain additional information. We can also determine the presence of restriction site in the template DNA by subjecting the PCR product to re­striction endonuclease prior to electro­phoresis.

    This protocol is a type of restric­tion fragment length polymorphism (RFLP) analysis which has immense significance in the construction of genome maps and studying genetic diseases. Electrophoretic analysis of PCR product can also help us in identifying the insertion or de­letion mutation in the amplified region.

    (b) Cloning of PCR Products:

    During the cloning experiment many times we take the help of PCR directly. If the gene of in­terest is in a very less quantity, then we need to amplify it. This is done by the help of PCR which produces enough copies of our target DNA so that we can afford to start the experiment.

    (c) Sequencing of PCR Products:

    The se­quencing of PCR product is done by the help of an automated sequencer machine. There is no need for radio-la­belling and autoradiography. This is an im­proved way to sequence DNA because of its speed and because it can be analysed by computer rather than a person.

    PCR has a number of applications especially where speed and the number of samples to be processed are important or where the amount of DNA available is very limited. Here are some of the applications.

    A. DNA Sequencing:

    PCR in the presence of di-deoxynucleoside triphosphates (ddNTPs), used for DNA sequencing, al­lows DNA sequencing reactions to be run successfully with very small amounts of template.

    B. Diagnostic:

    PCR is useful as a diagnostic tool, e.g., in the identification of specific genetic traits or for the detection of patho­gens or food contaminants. One of the first applications of PCR to genetic diagnosis was for sickle cell anaemia.

    C. Forensic:

    The ability to amplify DNA from regions of the genome that are highly polymorphic (and which are variable be­tween individuals) starting with samples containing very small amounts of DNA (e.g., single hairs or traces of body fluids, such as blood and semen) leads to applica­tions in forensic work.

    D. Present-Day Population Genetics:

    It al­lows for the determination of frequencies of particular alleles in a large collection of individuals. A particular advantage of us­ing PCR in population genetic studies is that, with appropriately designed specific primers, it may be possible to amplify DNA from one organism that cannot be sepa­rated from others, such as a particular bacterial strain in a mixed population.

    (Such primers will anneal to the target DNA from the organism of interest, but not to DNA from others.)

    E. Archaeology and Evolution:

    PCR can be used with old material as well as more re­cent samples, and it is often possible to amplify ancient DNA from museum speci­mens and archaeological remains. Mostly mitochondrial DNA or chloroplast DNA is used. This allows inferences to be made about the origins of particular populations or species.

    7. Precautions and Drawbacks:

    I. Size:

    The size of fragments that can be am­plified is limited by the processivity of the polymerase used. Using a mix­ture of polymerases that includes a proof­reading enzyme increases the size of pro­duct that can be obtained (up to 10 kbp or more), because incorrectly incorporated nucleotides can be removed rather than causing chain termination.

    Ii. Amplifying the Wrong Sequence:

    PCR depends on the ability of the primers to anneal to the correct sequence, and this depends on the conditions of annealing (ionic concentration, temperature, etc.) and the actual sequence (or sequences if mixed sites are included) of the primers.

    It is possible for primers to anneal to the “wrong” part of the target DNA, through chance complementarity. If this happens and the primers anneal in the correct ori­entation to each other (i.e., directing syn­thesis towards each other) and at sites that are not too far apart, then the result is the amplification of a sequence other than the desired one.

    The possibility of incorrect annealing may be avoided by use of longer primers, which will be more specific in their annealing sites. Raising the temperature and adjust­ing the concentration of magnesium ions (which stabilize primer-template binding) can be used to increase the specificity of primer binding.

    Iii. Contamination:

    Because of the extraor­dinary sensitivity of PCR, there is a par­ticular danger of contaminating the DNA sample to be amplified with extraneous material. This is particularly important when using material containing only small amounts of DNA, as with archaeological work.

    Contamination might be of labora­tory origin (e.g., from aerosols created by pipetting solutions containing related DNA sequences, including material amplified previously by PCR) or of external ori­gin (perhaps by bacterial, fungal or human contamination of sample tissue).

    Laboratory contamination can be mini­mized by precautions such as careful use and design of pipettes, separation of the pre-PCR and post-PCR stages of an experi­ment into different rooms. Contamination from other sources can be reduced by care­ful handling and preparation of a sample before amplification.

    Iv. Sequence Heterogeneity:

    Amplification may give rise to a mixture of molecules of slightly different sequences.

    A mixture could arise for several reasons:

    If the template DNA came from an individual heterozygous at the locus in question, each of the alleles present should be represented in similar quantities in the PCR prod­ucts.

    (b) Population Heterogeneity:

    If the tem­plate DNA came from several individu­als rather than a single one, heteroge­neity in the population may give rise to heterogeneity in the products.

    (c) DNA Damage and Polymerase Error:

    Heterogeneity can also arise from damage to DNA before amplification, especially if the sample has not been carefully preserved. Therefore, this is particularly likely to be a problem with archaeological and forensic material.

    V. Jumping PCR:

    When degraded DNA is amplified, it may be that any given sample molecule is not long enough to span the entire distance between the two priming sites. The result in the first round of syn­thesis would be extension of the primer to the end of a fragmented molecule, but not all the way to the second primer site.

    How­ever, on a subsequent round of synthesis, the truncated amplification product may anneal to a different DNA fragment that contains the remaining region intact. This would then allow synthesis of the full PCR product. This is called jumping PCR. So it is sometimes possible to generate PCR products that are longer than any indi­vidual template molecule. This can be ad­vantageous when amplifying badly de­graded DNA.

    8. Modifications:

    A. Hot-Start PCR:

    As soon as the PCR re­agents have all been mixed together, it is possible for the DNA polymerase to start synthesis. This may happen while the re­action mixture is being heated for the first time, and is at a temperature low enough to allow non-specific annealing of primer to template, generating a range of non-spe­cific products.

    This problem would be pre­vented if DNA synthesis could not take place until the first cycle had reached its maximum temperature. This is the basis of hot-start PCR. In the simplest form, the DNA polymerase is not added to the reaction tubes until they have reached the DNA melting temperature of the first cycle. This is satisfactory where small numbers of samples are being processed, but not with large numbers.

    B. Touch-Down PCR:

    The annealing tem­perature used in conventional PCR is usu­ally several degrees below the maximum at which primers can remain bound to tem­plate, to ensure stable binding. However, this use of a lower temperature permits a small amount of mismatching between primers and template, which may allow primers to bind to incorrect sites and gen­erate spurious products.

    The effects of this can be reduced with touch-down PCR. In this, a high annealing temperature is used initially (at which even correct bind­ing may not be possible). The annealing temperature is reduced in subsequent rounds. There will, therefore, come a point at which correctly matched primer-tem- plate annealing is just possible, but incor­rect matching is not and the desired prod­ucts will be the most abundant.

    C. Nested PCR:

    Here, two successive PCRs are carried out. The first PCR uses a con­ventional template. The products of the first PCR are then used as the template for the second PCR, with primers that are designed to anneal within the desired prod­uct of the first PCR.

    Although the first PCR may generate some non-specific products in addition to the desired products, it is unlikely that the non-specific products will also contain annealing sites for both the primers used in the second PCR. Thus, only the desired products from the first PCR are likely to be suitable templates for the second.

    D. Inverse PCR:

    It is possible to arrange for the amplification of sequences outside the primers, in a technique called inverse PCR (IPCR). In this technique the sample DNA is first cut with an enzyme outside the region whose sequence is already known.

    The resulting linear molecules are then circularized, by ligation under condi­tions that favour intermolecular reactions. A second restriction digestion is then done, using an enzyme cutting within the region of known sequence.

    The result is now that the first fragment containing this sequence has been turned ‘inside out’, leaving known sequence on the outside and the material that had previously been flanking it within. Primers complementary to the known sequence on the outside of the mole­cule can now be used to amplify the region of interest between them.

    E. Reverse Transcriptase PCR:

    It is often convenient to amplify RNA molecules, per­haps as a precursor to cloning them, or to estimate the abundance of a particular mRNA in a sample. This is usually done by having a round of reverse transcription, using a reverse transcriptase enzyme and a single primer, to make a single strand of cDNA prior to the PCR itself.

    The primer for reverse transcription could be oligo-dT for general cDNA synthesis from polyadenylated messages, or it could be specific to a particular message.

    F. In Situ PCR:

    It is possible to carry out PCR using permeabilized tissue, such as thin sections on a microscope slide. This requires a specially adapted PCR machine to accommodate the slide. If the PCR prod­uct can be detected (perhaps by hybridiza­tion, also in situ), then this allows one to identify where in the tissue the target nucleic acid is located.

    G. Asymmetric PCR:

    By reducing the amount of one of the two primers, it is pos­sible to arrange for preferential amplifica­tion of one of the strands, resulting in a preparation of single-stranded DNA, which has a number of uses in molecular biology. Preferential amplification of one strand in this way is known as asymmet­ric PCR.

    H. Anchored PCR:

    Anchored PCR is applied when only one piece of sequence (and therefore, one priming site) for the region of interest is known. The aim is to attach the region to be amplified to a piece of known sequence and then to use that as the second priming site.

    There are two ways in which this can most easily be done. One is to fragment the sample DNA and ligate it to molecules of known sequence, such as a vector. This known sequence is used as the basis for designing one of the two PCR primers. The second method is to add tails enzymatically to the sample DNA or the molecules produced after the first round of synthesis.

    I. Emulsion PCR:

    In a conventional PCR, the reactions are carried out inside plastic tubes. It is possible to incorporate all the reagents inside lipid droplets and carry out PCR on a much smaller scale. This has certain advantages. It is possible to in­crease and decrease the temperature of small droplets very quickly.

    In addition, if each droplet contains a single template molecule at the start, then all the products in an individual droplet result from the am­plification of a single template molecule. The method is also called droplet PCR.

    J. Isothermal Amplification:

    The repeated heating and cooling required by PCR lim­its how quickly the process can be carried out. Loop-mediated isothermal ampli­fication (LAMP) has been developed, which allows templates to be amplified at a constant temperature (typically around 65°C).

    It uses a DNA polymerase with strand-displacing activity and avoids the need for heating to high temperatures. This method is used for the detection of pathogens outside of specialist laboratories.

    K. Real Time PCR:

    It is possible to use PCR to estimate the abundance of a particular nucleic acid molecule in a sample. This can be done by real time PCR. This can be done in two ways.

    In the first, a fluorescent, double-stranded DNA (dsDNA)-binding dye (such as SYBR green) is present in the PCR. As dsDNA prod­uct accumulates, the amount of fluorescence from the dye increases, and this can be de­tected.

    The experiment requires a PCR ma­chine that is also equipped with a fluorescence measurement facility. Because the method simply detects dsDNA, it measures the amount of PCR product at a given time regardless of whether it is from the correct region.

    The second approach to real-time PCR al­lows detection of a specific product, rather than dsDNA in general, and uses a specially syn­thesized probe oligonucleotide. This probe is designed to anneal within the region to be amplified and carries a fluorescent reporter dye at one end and a quencher at the other end of the molecule.

    If the quencher and the reporter are in close proximity (i.e., attached to the same oligonucleotide), then the quencher stops the reporter from fluorescing.

    During PCR, the probe will anneal to single-stranded DNA within the target region. When the poly­merase meets the annealed probe, the 5′-3′ exonuclease activity of the enzyme degrades the probe, liberating the reporter from the quencher. Thus, the fluorescent reporter ac­cumulates during the course of the PCR. This type of PCR mechanism is shown in the dia­gram given above.


    PCR (polymerase chain reaction)

    Let's say you have a biological sample with trace amounts of DNA in it. You want to work with the DNA, perhaps characterize it by sequencing, but there isn't much to work with. This is where PCR comes in. PCR is the amplification of a small amount of DNA into a larger amount. It is quick, easy, and automated. Larger amounts of DNA mean more accurate and reliable results for your later techniques.

    PCR can be used to create a DNA "fingerprint," which is unique to each individual. These DNA fingerprints can be useful in real-world applications relating to paternity/maternity, kinship, and forensic testing.

    The technique was developed by Nobel laureate biochemist Kary Mullis in 1984 and is based on the discovery of the biological activity at high temperatures of DNA polymerases found in thermophiles (bacteria that live in hot springs).

    Most DNA polymerases (enzymes that make new DNA) work only at low temperatures. But at low temperatures DNA is tightly coiled, so the polymerases don't stand much of a chance of getting at most parts of the molecules.

    But these thermophile DNA polymerases work at 100C, a temperature at which DNA is denatured (in linear form). This thermophilic DNA polymerase is called Taq polymerase, named after Thermus aquaticus, the bacteria it is derived from.

    Taq polymerase, however, has no proofreading ability. Other thermally stable polymerases, such as Vent and Pfu, have been discovered to both work for PCR and to proofread.

    You'll need four things to perform PCR on a sample:

    1. The target sample. This is the biological sample you want to amplify DNA from.

    2. A primer. Short strands of DNA that adhere to the target segment. They identify the portion of DNA to be multiplied and provide a starting place for replication.

    3. Taq polymerase. This is the enzyme that is in charge of replicating DNA. This is the polymerase part of the name polymerase chain reaction.

    4. Nucleotides. You'll need to add nucleotides (dNTPs) so the DNA polymerase has building blocks to work with.

    There are three major steps to PCR and they are repeated over and over again, usually 25 to 75 times. This is where the automation is most appreciated.

    1. Your target sample is heated. This denatures the DNA, unwinding it and breaking the bonds that hold together the two strands of the DNA molecule, leaving you with single stranded DNA (ssDNA).

    2. Temperature is reduced and the primer is added. The primer molecules now have the opportunity to bind (anneal) to the pieces of ssDNA. This labels the portions of DNA to be amplified and provides a starting place for replication.

    3. New pieces of ssDNA are made. Taq polymerase catalyzes the generation of new pieces of ssDNA that are complimentary to the portions marked by the primers. The job of Taq polymerase is to move along the strand of DNA and use it as a template for assembling a new strand that is complimentary to the template. This is the chain reaction in the name polymerase chain reaction.

    PCR is so efficient because it multiplies the DNA exponentially for each of the 25 to 75 cycles. A cycle takes only a minute or so and each new segment of DNA that is made can serve as a template for new ones.

    Perhaps the most important thing to remember is to be very aware of contamination. If, for example, you unknowingly slough off a piece of skin into your sample, then your DNA may be amplified in the PCR reaction. Here are some other factors to optimize your results with PCR:

    1. Annealing temperature. Starts at the low end of what you think will work, then move up as necessary. If the temperature is too low, the primers will make more mistakes and you'll get too many bands when you run your sample on a gel. If the temperature is too high you will get no results and your gel will be blank. You want to be about 3C to 5C below the melting temperature (Tm). A rough formula for determining Tm is Tm=4(G + C) + 2(A + T).

    2. Magnesium concentration. You want your Mg2+ concentration to be about 1.5mM to 3mM. If you go too high, the polymerase will make more mistakes.

    3. Think carefully about primer design. Both primers should have approximately the same Tm so they both anneal at the same temperature. Two out of three bases on the 3' end should be G or C to get good hybridization (G and C have three H-bonds so you get better polymerization). Lastly, avoid primer dimers, which occur when the primers have ends that will anneal to each other. This will produce NO product.

    4. More is not necessarily better. More polymerase produces more nonspecific product, so don't just carelessly dump in a bunch of polymerase. Additionally, PCR reactions don't work if there is too much DNA.

    Taq polymerase does not work on RNA samples, so PCR cannot be used to directly amplify RNA molecules. The incorporation of the enzyme reverse transcriptase (RT), however, can be combined with traditional PCR to allow for the amplification of RNA molecules. After you add your RNA sample to the PCR machine, add a DNA primer as usual and allow it to anneal to your target molecule. Then add RT along with dNTPs, which will elongate the DNA primer and make a cDNA copy of the RNA molecules and run the PRC reaction as usual. The product of RT-PCR is a double stranded DNA molecule analogous to the target segment of the RNA molecule.