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I amplified my DNA with real time PCR. The amplification curve shows good amplification but not a single band is observed in my gel after gel electrophoresis. What would be the possible reasons?
I am attaching a photograph of my gel below. My ladder opened correctly but not a single band can be observed:
As you can see from the bottom of the gel, the brightness of the bands have reduced even for the DNA ladder/marker. The main reason for this is that when you are running an agarose gel containing EtBr, due to the positive charge of the EtBr, the stain runs to the negative chamber, against the direction of the DNA migration as @Chris has pointed out! Although some of the DNA will stain but the rest of the EtBr will migrate towards the top of the gel (where the DNA is loaded).
A quick remedy for this is once you ran your gel is to soak your gel in a separate container containing your stain and running buffer such as TAE. I do this for one hour and that makes a huge difference!! Also make sure you regularly change the buffer in your agarose gel running apparatus and don't use the same buffer for a long time since again as @Chris has correctly pointed out there is a reduced conductivity in the older buffers when they heat up so migration does not work as well! Obviously this response assumes that you have a good degree (amount) of DNA in your gel bands, which can be calculated by nanodrop once you extracted the DNA using an extraction kit, or whatever method you normally use. I hope this helps!
This answer is more suited to the title of the question since after reading the main question a bit more carefully I'm slightly confused! So do you see anything at all or your product is far too faint? could you tell us where on the gel, relative to the ladder you expect to see this band? are they the ones at the button of the gel, below the loading buffer?
Troubleshooting PCR Part Three: Solutions for Weak Bands and Smearing
When doing PCR, you can encounter all sorts of issues, which is why we have been doing a series on troubleshooting tips for different obstacles that occur. In the last two articles, I discussed nonspecific binding and what happens when you’re not getting any bands. In this article, I’ll go over some tips to help you when you’re encountering weak results and smearing.
Weak Results: When encountering weak results, there are a lot of simple troubleshooting techniques to explore. Some researchers will redo their PCR and have everything adjusted at once, while others will tweak one thing at a time to solve the problem. If you need to know exactly what is going wrong with your PCR, then it’s best to take it one step at a time.
1.Check your DNA template. Your concentration might be too low and increasing it could greatly improve your results.
2.Increase your cycle times – this is especially helpful if you’re still facing low template concentrations.
3.DNA degradation could also be the issue. You may want to check the DNA quality and re-isolate if necessary.
5.Use fresh reagents – contamination is often an issue. It might be a good idea to use fresh aliquots of your PCR material.
Smeared Bands: There are several factors that might cause smearing to occur, and we have some simple solutions to fix that.
1.Reduce your template – Having too much template seems to be the most common issue. Try to reduce your template to see if that improves your results.
2.Lower cycle times – This is especially helpful when your template concentration is higher. Keep within 20-35 cycles.
3.Reduce extension times / Raise annealing temperature – both of these will help improve your PCR results by reducing the occurrence of nonspecific binding and smearing bands.
4.Change your TAE – For small gels, be sure you’re changing your TAE with every run. It’s understandable that some larger rigs can go a few runs without being changed. But this could be one of your issues.
5.Use fresh reagents – grab new aliquots because you might be encountering contamination that is causing degradation.
After taking a look at our series of articles, you may not have to rely so much on the PCR good luck charms and superstition to get you through the task.
"To understand the universe is to understand math." My 8th grade
math teacher's quote meant nothing to me at the time. Then came
college, and the revelation that the adults in my past were right all
along. But since math feels less tangible, I fell for biology and have
found pure happiness behind my desk at GoldBio, learning, writing
and loving everything science.
How do you interpret the results of PCR reactions from electrophoresis?
I'm in a Biology 101 class for my General Education Requirements (I'm a non-science major), and I'm having a hard time understanding exactly what I'm looking at when it comes to PCR Results.
Included is an image of the results: http://imgur.com/BSo0u1T
Band 1 is the Ladder, Band 2 is the Experimental, Band 3 is the Negative Control, and Band 4 is the Positive Control.
The experiment was an attempt to amplify a specific oncogene in the genome of tumor cells. Band 2 is the DNA Sample from the tumor cells.
What is the point of the ladder? What exactly am I looking at when it comes to the results here?
I'm really confused, and my teacher is of no help. Iɽ appreciate any help you could give me!
since you are a non-science major, I'll try to keep this short and to the point:
I'll assume you know what PCR is and does (other people on here have given good descriptions, if you need)
electrophoresis separates out DNA based on fragment size using an electric charge (DNA is slightly negative, so it moves towards the positive charge). The smaller the fragment, the farther it moves (smaller fragments have less ɽrag'). Since the DNA starts in the wells at the top, the lower the band is on the gel, the smaller in size it is.
The ladder is just to verify the size of the fragments in your sample. You should know what size each of the bands are in your ladder, depending on what ladder you used, and compare that to your sample to determine the size of the sample fragments.
Negative control is blank, and that is good. Just ignore it for now.
Your positive control shows the size of the DNA fragment you're interested in. Now compare that to your experimental sample. see how there is a band in your sample that is the same size as the positive control? That means your experimental sample probably contained the specific oncogene you were looking for (you would have to sequence it or do another test to be completely 100% positive).
The other smaller but brighter band could be many things. maybe a different version of the gene or some non-target gene amplification. In any case, just ignore it for this problem. The most important part is that there is a band in your sample that is the same size as the positive control, so you can say the experimental sample most likely contains the specific oncogene.
Hope this helps! Feel free to ask questions
PCR amplifies a specific section of DNA, when you run these DNA fragments on an agarose gel you exploit the fact that different lengths of DNA can move through the gel at different speeds (the longer the length the slower it goes).
The ladder is a pre-determined set of base pairs of different known length, which is necessary because the distance that the DNA travels is dependent on many fragments so you need a reference to compare it to. Okay you may be able to identify a longer or shorter sequence without it, but ladders allow you to be more specific about the length. This is especially important if you're amplifying something whose length you already know, such as the DNA of a protein or tumour marker. You will know its size in the genome so you can identify your fragment in the gel knowing that you expect it at 750bp at the ladder shows you have a band on your sample at approximately that length.
Now for your experiment lane 1 is the ladder, and you should be able to identify what each band means by looking up the information from the name of the ladder (eg, NEB 1kbp DNA ladder) and comparing it to their reference example. This is for the sake of accuracy, but if you have known controls then you should generally be able to identify it without this (and I think it is likely enough for your gen-ed reqs). To orient yourself the top of the gel is where you place your sample, meaning the lower the band on the image, the smaller the length of DNA.
Getting to the experimental lanes, you can see that lane 3 is blank for your neg control, this is good. Lane 4 is the positive control, meaning this is where you should expect to see your band, if successful, in your experiment. Now for the experiment lane you can probably see a faint band around the level of the positive control, but a stronger band further down. This indicates that although you cloned a fragment at a similar length to your positive control, something didn't quite go right because you have more DNA with a smaller length. In addition it should be noted that although the top band is of similar length, this does not necessarily mean that it is your amplified marker, since technically you could have amplified something of a similar length from elsewhere in the genome.
For comparison here is a gel I ran. Lane 1 is DNA ladder, lane 2 is the experiment and lane 3 is the negative control. You see the strong bands at the top in the third lane, these indicate the unamplified genomic DNA, because it's so large in size it didn't (or couldn't) move much in the gel, whereas the second lane which is my experiment shows a single band of appropriate base pairs in length and no other bands (meaning it only amplified a region of that length and nothing else).
As for what happened and why, well it generally the biggest cause is a poorly designed primer (these are a short collection of nucleotides that bind to the corresponding area of DNA and help to initiate the cloning, they basically identify what part of the DNA is going to get cloned), or procedural errors during the experiment. If you get multiple bands in a PCR experiment it's usually a sign that your primer annealed to more than one area of the genome. When designing primers there are a number of factors you need to take into account. Since these are of short length,
20 base pairs, if you don't design them with specificity to your target area they may amplify other things as well. This is why we tend to use tools like Primer-BLAST which, after entering a primer sequence, helps to identify what sequences in the genome will be amplified, to double check that you only get what you want. This also helps to design your primers with specific criteria such as including an intron in the primer (which prevents you from amplifying mRNA, since this doesn't have introns only the genomic DNA should be amplified). Identifying the length of the smallest band in your DNA ladder can also give you clues as to the identity of that lower band in your experiment, and running primer-blast can help confirm this too (but I don't know how much of this is what you were given or actually did in class).
Now you do need to bear in mind what you are trying to achieve, I only wanted to clone a specific genetic sequence, whereas if you are working with a tumour cell there may inherently be changes in the genome of the cell so seeing something slightly different doesn't always mean your experiment went wrong. It could be that the tumour cell simply has mutations in the genome resulting in a smaller gene. As you can see a lot about science and experimentation is not only trying to get the damn thing to work but trying to understand and explain when things go wrong.
Hope this has helped, Iɽ be happy to answer any questions you have as well!
Molecular Analysis of Proteins
In many cases it may not be desirable or possible to study DNA or RNA directly. Proteins can provide species-specific information for identification as well as important information about how and whether a cell or tissue is responding to the presence of a pathogenic microorganism. Various proteins require different methods for isolation and characterization.
Polyacrylamide Gel Electrophoresis
A variation of gel electrophoresis, called polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins. In PAGE, the gel matrix is finer and composed of polyacrylamide instead of agarose. Additionally, PAGE is typically performed using a vertical gel apparatus (Figure 6). Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins based on their net charges. Alternatively, proteins can be denatured and coated with a negatively charged detergent called sodium dodecyl sulfate (SDS), masking the native charges and allowing separation based on size only. PAGE can be further modified to separate proteins based on two characteristics, such as their charges at various pHs as well as their size, through the use of two-dimensional PAGE. In any of these cases, following electrophoresis, proteins are visualized through staining, commonly with either Coomassie blue or a silver stain.
Figure 6. Click for a larger image. (a) SDS is a detergent that denatures proteins and masks their native charges, making them uniformly negatively charged. (b) The process of SDS-PAGE is illustrated in these steps. (c) A photograph of an SDS-PAGE gel shows Coomassie stained bands where proteins of different size have migrated along the gel in response to the applied voltage. A size standard lane is visible on the right side of the gel. (credit b: modification of work by “GeneEd”/YouTube)
Think about It
Clinical Focus: Karni, Part 3
This example continues Karni’s story that started in Microbes and the Tools of Genetic Engineering and above.
Figure 7. A bulls-eye rash is one of the common symptoms of Lyme diseases, but up to 30% of infected individuals never develop a rash. (credit: Centers for Disease Control and Prevention)
When Karni described her symptoms, her physician at first suspected bacterial meningitis, which is consistent with her headaches and stiff neck. However, she soon ruled this out as a possibility because meningitis typically progresses more quickly than what Karni was experiencing. Many of her symptoms still paralleled those of amyotrophic lateral sclerosis (ALS) and systemic lupus erythematosus (SLE), and the physician also considered Lyme disease a possibility given how much time Karni spends in the woods. Karni did not recall any recent tick bites (the typical means by which Lyme disease is transmitted) and she did not have the typical bull’s-eye rash associated with Lyme disease (Figure 7). However, 20–30% of patients with Lyme disease never develop this rash, so the physician did not want to rule it out.
Karni’s doctor ordered an MRI of her brain, a complete blood count to test for anemia, blood tests assessing liver and kidney function, and additional tests to confirm or rule out SLE or Lyme disease. Her test results were inconsistent with both SLE and ALS, and the result of the test looking for Lyme disease antibodies was “equivocal,” meaning inconclusive. Having ruled out ALS and SLE, Karni’s doctor decided to run additional tests for Lyme disease.
- Why would Karni’s doctor still suspect Lyme disease even if the test results did not detect Lyme antibodies in the blood?
- What type of molecular test might be used for the detection of blood antibodies to Lyme disease?
We’ll return to Karni’s example in later pages.
Steps in DNA Sample Processing
Following is a review of the steps involved in processing forensic DNA samples with STR markers. STRs are a smaller version of the VNTR sequences first described by Dr. Jeffreys. Samples obtained from crime scenes or paternity investigations are subjected to defined processes involving biology, technology, and genetics.
Following collection of biological material from a crime scene or paternity investigation, the DNA is first extracted from its biological source material and then measured to evaluate the quantity of DNA recovered. After isolating the DNA from its cells, specific regions are copied with a technique known as the polymerase chain reaction, or PCR. PCR produces millions of copies for each DNA segment of interest and thus permits very minute amounts of DNA to be examined. Multiple STR regions can be examined simultaneously to increase the informativeness of the DNA test.
The resulting PCR products are then separated and detected in order to characterize the STR region being examined. The separation methods used today include slab gel and capillary electrophoresis (CE). Fluorescence detection methods have greatly aided the sensitivity and ease of measuring PCR-amplified STR alleles. After detecting the STR alleles, the number of repeats in a DNA sequence is determined, a process known as sample genotyping.
The specific methods used for DNA typing are validated by individual laboratories to ensure that reliable results are obtained and before new technologies are implemented. DNA databases, such as the one described earlier in this chapter to match Montaret Davis to his crime scene, are valuable tools and will continue to play an important role in law enforcement efforts.
The resulting DNA profile for a sample, which is a combination of individual STR genotypes, is compared to other samples. In the case of a forensic investigation, these other samples would include known reference samples such as the victim or suspects that are compared to the crime scene evidence. With paternity investigations, a child's genotype would be compared to his or her mother's and the alleged father(s) under investigation. If there is not a match between the questioned sample and the known sample, then the samples may be considered to have originated from different sources. The term used for failure to match between two DNA profiles is 'exclusion.'
If a match or 'inclusion' results, then a comparison of the DNA profile is made to a population database, which is a collection of DNA profiles obtained from unrelated individuals of a particular ethnic group. For example, due to genetic variation between the groups, African-Americans and Caucasians have different population databases for comparison purposes.
Finally a case report or paternity test result is generated. This report typically includes the random match probability for the match in question. This random match probability is the chance that a randomly selected individual from a population will have an identical STR profile or combination of genotypes at the DNA markers tested.
Gel Loading Dye, Purple (6X)
Gel Loading Dye, Purple (6X) is the premier gel loading dye from NEB for sharp, tight bands.
- No UV Shadow
- Contains Ficoll ® for brighter, tighter bands
- Contains SDS for improved band sharpness
- Contains EDTA to stop enzymatic reactions
- Compatible with agarose and non-denaturing polyacrylamide gels
- Our Purple Gel Loading Dye sharpens bands and eliminates the UV shadow seen with other dyes. Available with or without SDS (NEB #B7025).
Gel Loading Dye, Purple (6X) is a pre-mixed loading buffer which contains a combination of two dyes, Dye 1 (pink/red) and Dye 2 (blue). The red dye serves as the tracking dye for both agarose and non-denaturing polyacrylamide gel electrophoresis. The two dyes separate upon gel electrophoresis the red band is the major indicator and migrates similarly to Bromophenol Blue on agarose gels. Specifically chosen, this dye does not leave a shadow under UV light. This solution contains SDS, which often results in sharper bands, as some restriction enzymes are known to remain bound to DNA following cleavage. EDTA is also included to chelate magnesium (up to 10 mM) in enzymatic reactions, thereby stopping the reaction. The dye also contains Ficoll, which creates brighter and tighter bands when compared to glycerol loading dyes. This product is packaged as 4x1 ml vials.
Attention SYBR® Safe and GelRed&trade dye users: Due to an increased concentration of SDS* in B7024S, NEB recommends using Gel Loading Dye, Purple, No SDS (6X) (NEB# B7025S) instead.
Comparison of dye fronts
Nucleic acid samples were run on an agarose gel, with either Purple or Blue loading dyes. Both dyes ran at the same speed. UV shadow comparison
The new Gel Loading Dye, Purple (6X) (Lane 1) included in the Quick-Load Purple 1kb DNA Ladder does not cast a UV shadow over the underlying bands, unlike the Gel Loading Dye, Blue (6X) (Lane 2).
Question: 1. You Will Estimate The Quantity Of DNA Present In The PUC18 DNA Prep By Gel Band Brightness. Compare Your Linear Plasmid Band In The HindIII Digest Lane To The λ-HindIII Marker. If, For Example, Your Linearized Plasmid Is Half As Bright As The 4, 361 Bp Band Of The Marker, Then Your Approximate DNA Quantity Is 90/2 Or 45 Ng. You Loaded 10 μl Of .
1. You will estimate the quantity of DNA present in the pUC18 DNA prep by gel band brightness. Compare your linear plasmid band in the HindIII digest lane to the λ-HindIII marker. If, for example, your linearized plasmid is half as bright as the 4, 361 bp band of the marker, then your approximate DNA quantity is 90/2 or 45 ng. You loaded 10 μl of the HindIII digest onto the gel so your original concentration of DNA in the digest is 45 ng/10 μl or 4.5 ng/μl. You put 10 μl of DNA in your digest. All together then, you have 4.5 ng/μl x 10 μl or 45 ng of DNA in the digest.
I am comparing my linear Hind III digest plasmid to Fragment ____ of the λ-HindIII digest.
The amount of DNA in the linearized plasmid is ________ and I reach this number because_________________________________________________________________.
The original concentration (μg/μl) of pUC18 plasmid DNA in the HindIII digest is __________________.
The total amount of pUC18 plasmid DNA in the HindIII digest is ______________μg.
The pBR322-BstNI digest cannot be used for quantitation but can be used to estimate fragment size. You may find this marker useful in analyzing your data (but quantitate the DNA using the lambda-HindIII digest marker).
Tube III: 1 μl HindIII, 1 μl ScaI
Tube IV: 1 μl BglI, 1 μl ScaI
This is an example of how to answer this question
I am comparing my linear HindII digest plasmid to Fragment 4 of the λ-HindIII digest. The amount of DNA in the linearized plasmid is 45 ng and I reach this number because the assume band looks about half as bright compared to Fragment 4 of the λ-HindIII digest. The original concentration of pUC18 plasmid in the HindIII is 45 ng/10 μl or 4.5 ng/μl. The total amount of pUC18 plasmid DNA in the HindIII digest is 4.5 ng (because I added 10 μl of DNA in the digest, so 4.5 ng/μl x 10μl = 45ng). Too much ethidium bromide was added into the solution, which cause about half of the gel to fluoresce. Therefore, I have to guess the sizes of the marker fragments, which means that this estimation is not very accurat
How DNA Works
DNA is a long molecule. For example, a typical bacterium, like E. coli, has one DNA molecule with about 3,000 genes (A gene is a specific sequence of DNA nucleotides that codes for a protein. We'll talk about this later). If drawn out, this DNA molecule would be about 1 millimeter long. However, a typical E. coli is only 3 microns long (3 one-thousandths of a millimeter).So to fit inside the cell, the DNA is highly coiled and twisted into one circular chromosome.
Complex organisms, like plants and animals, have 50,000 to 100,000 genes on many different chromosomes (humans have 46 chromosomes). In the cells of these organisms, the DNA is twisted around bead-like proteins called histones. The histones are also coiled tightly to form chromosomes, which are located in the nucleus of the cell. When a cell reproduces, the chromosomes (DNA) get copied and distributed to each offspring, or daughter, cell. Non-sex cells have two copies of each chromosome that get copied and each daughter cell receives two copies (mitosis). During meiosis, precursor cells have two copies of each chromosome that gets copied and distributed equally to four sex cells. The sex cells (sperm and egg) have only one copy of each chromosome. When sperm and egg unite in fertilization, the offspring have two copies of each chromosome (see How Sex Works).
How can I get brighter DNA bands? - Biology
Semi-Conservative, Conservative, & Dispersive models of DNA replication
In the semi-conservative model, the two parental strands separate and each makes a copy of itself. After one round of replication, the two daughter molecules each comprises one old and one new strand. Note that after two rounds, two of the DNA molecules consist only of new material, while the other two contain one old and one new strand.
In the conservative model, the parental molecule directs synthesis of an entirely new double-stranded molecule, such that after one round of replication, one molecule is conserved as two old strands. This is repeated in the second round.
In the dispersive model, material in the two parental strands is distributed more or less randomly between two daughter molecules. In the model shown here, old material is distributed symmetrically between the two daughters molecules. Other distributions are possible.
The semi-conservative model is the intuitively appealing model, because separation of the two strands provides two templates, each of which carries all the information of the original molecule. It also turns out to be the correct one (Meselson & Stahl 1958).
Click here to order our latest book, A Handy Guide to Ancestry and Relationship DNA Tests
Can a couple sire a baby that is significantly darker or lighter than either individual?
-A curious adult from North Carolina
The short answer is, yes! A couple can have a baby with a skin color that isn’t between their own. The long answer, though, is much more interesting.
The long answer has to do with the parts of your DNA that give specific instructions for one small part of you. In other words, your genes.
It turns out that there isn’t just one or even a few genes involved in skin color. There are hundreds of different stretches of DNA all working together that decide your skin color.
Some of these genes can have big effects while others fine-tune a final color. On top of all that, your actions can help change how your body reads your DNA! For example, staying out in the sun turns on genes that darken your skin.
Different Types of Genes Get Shuffled Around
Everyone has two copies of each gene, one from their mom and one from their dad. The copies are mostly the same.
Note the word mostly. If we all had the same DNA we’d all be identical twins!
Turns out that we are all unique because sometimes the copies of each gene are slightly different. Scientists call these slightly different genes “alleles”.
These genes and alleles are kind of like a deck of cards. Each gene is like a different card (ace, two, three… jack, queen, king). And, like cards that come in different suits (spades, clubs, diamonds, hearts), there's more than one version of each card.
Imagine your mom and dad give you one of each card from their own decks. You’ll get two jacks, but they might be of the same suit (two jacks of hearts) or different (one jack of hearts and one jack of spades). In genetics, the word for two of the same is homozygous, the word for one of each is heterozygous.
Now let’s pretend the black cards would lead to dark skin and the red cards would lead to light skin. If you have a dark dad (all clubs and spades) and a light mom (all hearts and diamonds) you will end up with one red card and one black card like this:
This mixed hand would give you medium toned skin.
If your partner has similar parents, then he or she will also end up with a mixed deck. Maybe something like this:
When you have a baby together, you and your partner will each give a random half of your cards to your baby.
Odds are the baby will get black and red cards. But it’s possible that your baby will get all red cards from both of you like this:
In this case, the baby would be much lighter than either of you.
The same logic applies to the black cards. If by chance your baby got mostly clubs and spades from you and your partner, then the baby would end up with much darker skin than either of you:
As you can see, if you have two babies, they might end up with very different decks! And so very different skin colors.
This random dealing actually happens. Some mixed race parents have twins that look very different (click here, here and here for some great pictures of real-life examples).
Some of these families answer your question: parents can have children with skin color that is significantly lighter or darker than their own.
Sometimes One Gene is Stronger than the Others
Sometimes a particular gene can have a much bigger effect than other genes. Scientists call this “different effect size.”
In the previous card example, we pretended that every card had the same value. Having a red queen had the same impact as a red two.
A more accurate game would be if each red number card had a different point value. A red queen would add a higher score than a red two. Some genes matter a lot and some just fine-tune the color.
To make the card game even more accurate, we can add one more rule: if you get two queens of hearts, you get an extra 1,000 points. Let’s see how that rule can impact things.
Imagine a slight variation on the two parents from before. In this case, your hand has one queen of hearts and your spouse’s hand has one queen of hearts like this:
You both have the around the same skin color as before because neither of you get the bonus points for two queens of hearts. But there’s a chance your kid will end up with two queens of hearts like this:
Here, even though the child has the same number of black and red cards as either parent, the child is much, much lighter than either parent because of those two queens of heart.
This may seem like a silly rule, but it’s actually how some genes work. In fact, it’s what happens for people with light skin and red hair.
There is a gene called MC1R that acts as an on switch for darker skin. Usually the sun is able to flick this switch and cause people to tan.
However, there is one type of this gene that doesn’t work. Like those two queens of hearts, if both of your copies of MC1R don’t work, you end up with way lighter skin. You score that 1,000 points!
When you need two copies of an allele to see a trait, this is called recessive. It takes two nonworking (recessive) MC1R alleles to give way lighter skin (the trait).
It turns out this switch doesn’t just change skin color. Hair color is also affected!
The rest of the person’s genes are saying, “make the hair colored!” so the person isn’t going to be blonde. But the person can’t flick the switch for brown, so their hair turns out red!
Genes Are Important, But You Still Have Control.
Your DNA contains all the information to make you. But that doesn’t mean it controls all of your future!
Genes, like MC1R, contain information about how a person will react to sun. Some people will burn while others will tan and still others will get covered in freckles. But each person can control how much he or she goes out into the sun.
Someone who likes to spend time in the sun will probably be darker than their parents who spend all their time indoors. Yes, they’re only tanner, but they could be significantly darker than their parents!