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My father died in 1987. I have an old cowboy hat of his. Can I use a FamilyTreeDNA test kit to recover any DNA?
Agarose Gel Electrophoresis
We use electrophoresis to separate nucleic acids or proteins by size and/or charge, depending on what we’ve put into our solutions (more of an issue with SDS-PAGE see Chapter 7 ). Basically, in gel electrophoresis, you put your samples into a gel matrix and your negatively charged nucleic acids (negatively charged due to their phosphate “backbone”) are repelled from the negative pole of your electrodes and toward the positive pole at the other end of the gel. Larger RNAs and DNAs (and proteins, in SDS-PAGE) have a harder time moving through the gel matrix and therefore you can separate your RNA/DNA/proteins by size. Additionally, this is the only way to check your results from end-point PCR (see PCR section in Chapter 4 ). In my experience, running electrophoresis gels is a balance between efficiency and beauty: running the gel at higher voltages will move the samples along the gel faster, but the resulting bands are more likely to be smeared (and if you set the voltage high enough, you’ll melt the gel and destroy all your hard work). Run the gels at lower voltages and you’ll get a prettier picture, but you’ll need to wait.
I have experience with three types of gel electrophoresis: agarose gel electrophoresis , denaturing (formaldehyde) gel electrophoresis, and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Agarose gels are cheap and can be used to examine RNA or DNA stability. Denaturing gel electrophoresis can be used with RNA for northern blotting, but is mostly unnecessary if your goal is to examine RNA stability, which can be examined using agarose gel electrophoresis. While one can make RNase-free solutions for agarose gel electrophoresis, I only find it necessary for the loading buffer and water but not the gel or buffer. RNase-free solutions are necessary if one chooses to perform northern blots, which are not discussed in this book. SDS-PAGE is most useful for separating proteins and is therefore discussed in Chapter 7 .
In agarose gel electrophoresis, one of two buffers is used: Tris-Acetate–EDTA (TAE) or Tris-Borate–EDTA (TBE). TBE has a higher buffering capacity than TAE. TBE buffer components precipitate out of solution when stored at higher concentrations (10× solution, for example), so I keep a 0.5× stock and avoid precipitation and stability issues.
Visualizing the results from agarose gel electrophoresis is typically performed one of two ways: including a DNA-binding fluorophore in the gel solution, or keeping that fluorophore in a buffer solution, washing the agarose gel in it, and using that solution for multiple gels. In both cases, you will use an ultraviolet light to excite the fluorophore and see the experiment’s results. The most common DNA-binding fluorophore is ethidium bromide (EtBr). EtBr is toxic and your local environmental health and safety board has recommendations for how to handle it do what they tell you to do. Another common fluorophore, and the one I use, is SYBR Safe™ 1 (Invitrogen). SYBR Safe™ does better on toxicity tests in lab animals than EtBr, but consuming a DNA-binding anything isn’t a good idea. In my experience, SYBR Safe™ degrades over time in free solution but not in its stored 10,000× concentrate. As such, I get consistent results by including SYBR Safe™ (or EtBr) in my gel solution after it has cooled enough, before casting the gel. When the fluorophore is free in solution, one needs to incubate gels for longer periods of time to get equivalent staining in reused solution however, if you’re examining gels for fluorescence, then you can’t tell if a lack of fluorescence is because the experiment didn’t work, you didn’t incubate in solution for long enough, or you need to make fresh fluorophore solution. Further, washing in fluorophore solution typically requires a wash in water to decrease the background noise, and you can leave it in water for so long that you wash out the fluorophore. In all, I think that including your fluorophore in the gel is the way to go, and you use less over time if you aren’t replacing your staining solution every couple of days.
A good reference for this section is the book At the Bench by Kathy Barker. 2
How DNA Evidence Works
For many years, fingerprints were the gold standard for linking suspects to a crime scene. Today, the gold standard is DNA evidence because DNA can be collected from virtually anywhere. Even a criminal wearing gloves may unwittingly leave behind trace amounts of biological material. It could be a hair, saliva, blood, semen, skin, sweat, mucus or earwax. All it takes is a few cells to obtain enough DNA information to identify a suspect with near certainty.
For this reason, law enforcement officials take unusual care at crime scenes. Police officers and detectives often work closely with laboratory personnel or evidence collection technicians to make sure evidence isn't contaminated. This involves wearing gloves and using disposable instruments, which can be discarded after collecting each sample. While collecting evidence, officers are careful to avoid touching areas where DNA evidence could exist. They also avoid talking, sneezing and coughing over evidence or touching their face, nose or mouth.
The following list shows some common sources of DNA evidence:
- A weapon, such as a baseball bat, fireplace poker or knife, which could contain sweat, skin, blood or other tissue
- A hat or mask, which could contain sweat, hair or dandruff
- A facial tissue or cotton swab, which could contain mucus, sweat, blood or earwax
- A toothpick, cigarette butt, bottle or postage stamp, all of which could contain saliva
- A used condom, which could contain semen or vaginal or rectal cells
- Bed linens, which could contain sweat, hair, blood or semen
- A fingernail or partial fingernail, which could contain scraped-off skin cells
When investigators find a piece of evidence, they place it in a paper bag or envelope, not in a plastic bag. This is important because plastic bags retain moisture, which can damage DNA. Direct sunlight and warmer conditions may also damage DNA, so officers try to keep biological materials at room temperature. They label the bags with information about what the material is, where it was found and where it will be transported. These are chain-of-custody procedures, which ensure the legal integrity of the samples as they move from collection to analysis.
Types DNA Evidence Analysis
DNA Typing — Polymerase Chain Reaction (PCR)
The evolution of DNA testing advanced significantly when Dr. Kary Mullis discovered that DNA could be copied in the laboratory much as it is in the natural world.
The copying process, known as polymerase chain reaction (PCR), uses an enzyme (polymerase) to replicate DNA regions in a test tube. By repeating the copying process, a small number of DNA molecules can be reliably increased up to billions within several hours.
RFLP analysis requires a biological sample about the size of a quarter, but PCR can be used to reproduce millions of copies of the DNA contained in a few skin cells. Since PCR analysis requires only a minute quantity of DNA, it can enable the laboratory to analyze highly degraded evidence for DNA. On the other hand, because the sensitive PCR technique replicates any and all of the DNA contained in an evidence sample, greater attention to contamination issues is necessary when identifying, collecting, and preserving DNA evidence. These factors may be particularly important in the evaluation of unsolved cases in which evidence might have been improperly collected or stored.
DNA Typing — Short Tandem Repeat (STR) Analysis
Short tandem repeat (STR) technology is a forensic analysis that evaluates specific regions (loci) that are found on nuclear DNA. The variable (polymorphic) nature of the STR regions that are analyzed for forensic testing intensifies the discrimination between one DNA profile and another. For example, the likelihood that any two individuals (except identical twins) will have the same 13-loci DNA profile can be as high as 1 in 1 billion or greater.
The Federal Bureau of Investigation (FBI) has chosen 13 specific STR loci to serve as the standard for CODIS. The purpose of establishing a core set of STR loci is to ensure that all forensic laboratories can establish uniform DNA databases and, more importantly, share valuable forensic information. If the forensic or convicted offender CODIS index is to be used in the investigative stages of unsolved cases, DNA profiles must be generated by using STR technology and the specific 13 core STR loci selected by the FBI.
More details on STR Analysis:
DNA Typing — Y-Chromosome Analysis
Several genetic markers have been identified on the Y chromosome that can be used in forensic applications. Y-chromosome markers target only the male fraction of a biological sample. Therefore, this technique can be very valuable if the laboratory detects complex mixtures (multiple male contributors) within a biological evidence sample. Because the Y chromosome is transmitted directly from a father to all of his sons, it can also be used to trace family relationships among males. Advancements in Y-chromosome testing may eventually eliminate the need for laboratories to extract and separate semen and vaginal cells (for example, from a vaginal swab of a rape kit) prior to analysis.
DNA Typing — Mitochondrial Analysis
Mitochondrial DNA (mtDNA) analysis allows forensic laboratories to develop DNA profiles from evidence that may not be suitable for RFLP or STR analysis. While RFLP and PCR techniques analyze DNA extracted from the nucleus of a cell, mtDNA technology analyzes DNA found in a different part of the cell, the mitochondrion (see exhibit 1). Old remains and evidence lacking nucleated cells — such as hair shafts, bones, and teeth — that are unamenable to STR and RFLP testing may yield results if mtDNA analysis is performed. For this reason, mtDNA testing can be very valuable to the investigation of an unsolved case. For example, a cold case log may show that biological evidence in the form of blood, semen, and hair was collected in a particular case, but that all were improperly stored for a long period of time.
Although PCR analysis sometimes enables the crime laboratory to generate a DNA profile from very degraded evidence, it is possible that the blood and semen would be so highly degraded that nuclear DNA analysis would not yield a DNA profile. However, the hair shaft could be subjected to mtDNA analysis and thus be the key to solving the case. Finally, it is important to note that all maternal relatives (for example, a person's mother or maternal grandmother) have identical mtDNA. This enables unidentified remains to be analyzed and compared to the mtDNA profile of any maternal relative for the purpose of aiding missing persons or unidentified remains investigations. Although mtDNA analysis can be very valuable to the investigation of criminal cases, laboratory personnel should always be involved in the process.
The risk of contamination of any crime scene can be reduced by limiting incidental activity. It is important for all law enforcement personnel at the crime scene to make a conscious effort to refrain from smoking, eating, drinking, littering or any other actions which could compromise the crime scene. Because DNA evidence is more sensitive than other types of evidence, law enforcement personnel should be especially aware of their actions at the scene to prevent inadvertent contamination of evidence.
The chain of custody of evidence is a record of individuals who have had physical possession of the evidence. Documentation is critical to maintaining the integrity of the chain of custody. Maintaining the chain of custody is vital for any type of evidence. In addition, if laboratory analysis reveals that DNA evidence was contaminated, it may be necessary to identify persons who have handled that evidence.
In processing the evidence, the fewer people handling the evidence, the better. There is less chance of contamination and a shorter chain of custody for court admissibility hearings.
Extracting DNA from Cells
To perform DNA fingerprinting, you must first have a DNA sample! In order to procure this, a sample containing genetic material must be treated with different chemicals. Common sample types used today include blood and cheek swabs.
These samples must be treated with a series of chemicals to break open cell membranes, expose the DNA sample, and remove unwanted components – such as lipids and proteins – until relatively pure DNA emerges.
PCR Amplification (Optional)
If the amount of DNA in a sample is small, scientists may wish to perform PCR – Polymerase Chain Reaction – amplification of the sample.
PCR is an ingenious technology which essentially mimics the process of DNA replication carried out by cells. Nucleotides and DNA polymerase enzymes are added, along with “primer” pieces of DNA which will bind to the sample DNA and give the polymerases a starting point.
PCR “cycles” can be repeated until the sample DNA has been copied many times in the lab if necessary.
Treatment with Restriction Enzymes
The best markers for use in quick and easy DNA profiling are those which can be reliably identified using common restriction enzymes, but which vary greatly between individuals.
For this purpose, scientists use repeat sequences – portions of DNA that have the same sequence so they can be identified by the same restriction enzymes, but which repeat a different number of times in different people. Types of repeats used in DNA profiling include Variable Number Tandem Repeats (VNTRs), especially short tandem repeats (STRs), which are also referred to by scientists as “microsatellites” or “minisatellites.”
Once sufficient DNA has been isolated and amplified, if necessary, it must be cut with restriction enzymes to isolate the VNTRs. Restriction enzymes are enzymes that attach to specific DNA sequences and create breaks in the DNA strands.
In genetic engineering, DNA is cut up with restriction enzymes and then “sewn” back together by ligases to create new, recombinant DNA sequences. In DNA profiling, however, only the cutting part is needed. Once the DNA has been cut to isolate the VNTRs, it’s time to run the resulting DNA fragments on a gel to see how long they are!
Gel electrophoresis is a brilliant technology that separates molecules by size. The “gel” in question is a material that molecules can pass through, but only at a slow speed.
Just as air resistance slows a big truck more than it does a motorcycle, the resistance offered by the electrophoresis gel slows large molecules down more than small ones. The effect of the gel is so precise that scientists can tell exactly how big a molecule is by seeing how far it moves within a given gel in a set amount of time.
In this case, measuring the size of the DNA fragments from the sample that has been treated with a restriction enzyme will tell scientists how many copies of each VTNR repeat the sample DNA contains.
It’s called “electrophoresis” because, to make the molecules move through the gel, an electrical current is applied. Because the sugar-phosphate backbone of the DNA has a negative electrical charge, the electrical current tugs the DNA along with it through the gel.
By looking at how many DNA fragments the restriction enzymes produced and the sizes of these fragments, the scientists can “fingerprint” the DNA donor.
Transfer onto Southern Blot
Now that the DNA fragments have been separated by size, they must be transferred to a medium where scientists can “read” and record the results of the electrophoresis.
To do this, scientists treat the gel with a weak acid, which breaks up the DNA fragments into individual nucleic acids that will more easily rub off onto paper. They then “blot” the DNA fragments onto nitrocellulose paper, which fixes them in place.
Treatment with Radioactive Probe
Now that the DNA is fixed onto the blotting paper, it is treated with a special probe chemical that sticks to the desired DNA fragments. This chemical is radioactive, which means that it will create a visible record when exposed to X-ray paper.
This method of blotting DNA fragments onto nitrocellulose paper and then treating it with a radioactive probe was discovered by a scientist name Ed Southern – hence the name “Southern blot.”
Amusingly, the fact that the Southern blot is named after a scientist and not the direction “south” did not stop scientists from naming similar methods “northern” and “western” blots in honor of the Southern blot.
X-Ray Film Exposure
The last step of the process is to turn the information from the DNA fragments into a visible record. This is done by exposing the blotting paper, with its radioactive DNA bands, to X-ray film.
X-ray film is “developed” by radiation, just like camera film is developed by visible light, resulting in a visual record of the pattern produced by the person’s DNA “fingerprint.”
To ensure a clear imprint, scientists often leave the X-ray film exposed to the weakly radioactive Southern blot paper for a day or more.
Once the image has been developed and fixed to prevent further light exposure from changing the image, this “fingerprint” can be used to determine if two DNA samples are the same or similar!
DNA from sweat on the hat band? - 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).
How does DNA Fingerprinting Work?
People everywhere expected the new millennium to bring surprises. But the particular shock and horror that rippled through the international viticulture community in 2000 was most unexpected. It had been found that sixteen of the most highly prized varieties of wine-making grapes were the products of mating between the classic Pinot and the classically undervalued Gouais grape.
This blew the proverbial cork off the industry's bottle because the Gouais was considered such an inferior specimen that there were even attempts to ban its cultivation in France during the Middle Ages. This proves that humble origins can still produce superior quality. More practically, though, knowledge of their heritage allows improved breeding of highly desirable subspecies of grape. And viticulturists everywhere had DNA fingerprinting technology to thank.
DNA fingerprinting is a term that has been bandied about in the popular media for many years, largely due to its power to condemn and save, but what does it involve? In short, it is a technique for determining the likelihood that genetic material came from a particular individual or group. 99% of human DNA is identical between individuals, but the 1% that differs enables scientists to distinguish identity. In the case of the grapes, scientists compared the similarities between different species and were able to piece together parent subspecies that could have contributed to the present prize-winning varieties.
The DNA alphabet is made up of four building blocks - A, C, T and G, called base pairs, which are linked together in long chains to spell out the genetic words, or genes, which tell our cells what to do. The order in which these 4 DNA letters are used determines the meaning (function) of the words, or genes, that they spell.
But not all of our DNA contains useful information in fact a large amount is said to be "non-coding" or "junk" DNA which is not translated into useful proteins. Changes often crop up within these regions of junk DNA because they make no contribution to the health or survival of the organism. But compare the situation if a change occurs within an essential gene, preventing it from working properly the organism will be strongly disadvantaged and probably not survive, effectively removing that altered gene from the population.
Left - DNA fingerprints from 6 different people, 1 in each lane (column).
DNA can be cut into shorter pieces by enzymes called "restriction endonucleases". The pieces of DNA can then be separated according to their size on a gel.
Each piece of DNA forms a band (the white lines on the gel). The smallest pieces travel the furthest and are therefore closest to the bottom of the gel. The larger pieces travel shorter distances and are closer to the top.
For this reason, random variations crop up in the non-coding (junk) DNA sequences as often as once in every 200 DNA letters. DNA fingerprinting takes advantage of these changes and creates a visible pattern of the differences to assess similarity.
Stretches of DNA can be separated from each other by cutting them up at these points of differences or by amplifying the highly variable pieces. 'Bands' of DNA are generated the number of bands and their sizes give a unique profile of the DNA from whence it derived. The more genetic similarity between a person - or grape - the more similar the banding patterns will be, and the higher the probability that they are identical.
In the non-coding regions of the genome, sequences of DNA are frequently repeated giving rise to so-called VNTRs - variable number tandem repeats. The number of repeats varies between different people and can be used to produce their genetic fingerprint. In the simple example shown above, person A has only 4 repeats whilst person B has 7. When their DNA is cut with the restriction enzyme Eco RI, which cuts the DNA at either end of the repeated sequence (in this example), the DNA fragment produced by B is nearly twice as big as the piece from A, as shown when the DNA is run on a gel (right). The lane marked M contains marker pieces of DNA that help us to determine the sizes. If lots of pieces of DNA are analysed in this way, a 'fingerprint' comprising DNA fragments of different sizes, unique to every individual, emerges.
But why bother? After all, I know where my wine comes from - Tesco's, right? Well, there are many relevant applications of DNA fingerprinting technology in the modern world, and these fall into three main categories: To find out where we came from, discover what we are doing at the present, and to predict where we are going.
In terms of where we came from, DNA fingerprinting is commonly used to probe our heredity. Since people inherit the arrangement of their base pairs from their parents, comparing the banding patterns of a child and the alleged parent generates a probability of relatedness if the two patterns are similar enough (taking into account that only half the DNA is inherited from each parent), then they are probably family. However, DNA fingerprinting cannot discriminate between identical twins since their banding patterns are the same. In paternity suits involving identical twins - and yes, there have been such cases - if neither brother has an alibi to prove that he could not have impregnated the mother, the courts have been known to force them to split child care costs. Thankfully there are other, less "Jerry Springer-esque", applications that teach us about our origins. When used alongside more traditional sociological methodologies, DNA fingerprinting can be used to analyse patterns of migration and claims of ethnicity.
DNA Fingerprinting can also tell us about present-day situations. Perhaps best known is the use of DNA fingerprinting in forensic medicine. DNA samples gathered at a crime scene can be compared with the DNA of a suspect to show whether or not he or she was present. Databases of DNA fingerprints are only available from known offenders, so it isn't yet possible to fingerprint the DNA from a crime scene and then pull out names of probable matches from the general public. But, in the future, this may happen if DNA fingerprints replace more traditional and forgeable forms of identification. In a real case, trading standards agents found that 25% of caviar is bulked up with roe from different categories, the high class equivalent of cheating the consumer by not filling the metaphorical pint glass all the way up to the top. DNA fingerprinting confirmed that the 'suspect' (inferior) caviar was present at the crime scene.
|In the example shown on the left, DNA collected at the scene of a crime is compared with DNA samples collected from 4 possible suspects. The DNA has been cut up into smaller pieces which are separated on a gel. The fragments from suspect 3 match those left at the scene of the crime, betraying the guilty party.|
Finally, genetic fingerprinting can help us to predict our future health. DNA fingerprinting is often used to track down the genetic basis of inherited diseases. If a particular pattern turns up time and time again in different patients, scientists can narrow down which gene(s), or at least which stretch(es) of DNA, might be involved. Since knowing the genes involved in disease susceptibility gives clues about the underlying physiology of the disorder, genetic fingerprinting aids in developing therapies. Pre-natally, it can also be used to screen parents and foetuses for the presence of inherited abnormalities, such as Huntington's disease or muscular dystrophy, so appropriate advice can be given and precautions taken as needed.
Acknowledgement: This article was co-authored with Dr Chris Smith, who also compiled the images.
Meselson and Stahl Experiment Steps
Meselson and Stahl performed a series of an experiment, which includes the following steps:
- Growth of E.coli: First, the E.coli were grown in the medium containing 15 NH4Cl for several generations. NH4 provides the nitrogen as well as a protein source for the growth of the E.coli. Here, the 15 N is the heavy isotope of nitrogen.
- Incorporation of 15 N: After several generations of E.coli, Meselson and Stahl observed that the 15 N heavy isotope has incorporated between the DNA nucleotides in E.coli.
- Transfer of E.coli cells: The DNA of E.coli labelled with 15 N isotope were transferred to the medium containing 14 NH4Cl. Here, the 14 N is the light isotope of nitrogen. The E.coli cells were again allowed to multiply for several generations. The E.coli cells will multiply every 20 minutes for several generations.
- Processing of DNA: For the processing or separation of DNA, the E.coli cells were transferred to the Eppendorf tubes. After that, caesium chloride is added, having a density of 1.71 g/cm 3 (the same of DNA). Finally, the tubes were subjected to high-speed centrifugation 140,000 X g for 20 hours.
After centrifugation, the DNA separates based on mass or density. Different DNA bands like heavy, intermediate and light DNA forms as a result of the concentration gradient created by CsCl.
The light DNA will consist of a pure 14 N isotope. An intermediate DNA band will indicate the combination or mixture of both 15 N and 14 N isotopes. The occurrence of heavy DNA bands will consist of a pure 15 N isotope.
The result, after two generations of E.coli, the following results were obtained:
In the F-1 generation: According to the actual observations, two DNA strands (with a mixture of both 15 N and 14 N isotopes) will produce in F-1 gen. The above diagram shows that the semiconservative and dispersive model obeys the pattern of growth explained by Meselson and Stahl.
Thus, it is clear that the DNA does not replicate via “Conservative mode”. According to the conservative model, the DNA replicates to produce one newly synthesized DNA and one parental DNA. Therefore, the conservative model was disapproved, as it does not produce hybrid DNA in the F-1 generation.
In the F-2 generation: According to the actual observation, four DNA strands (two with hybrid and the remaining two with light DNA) will produce in the F-2 generation. The hybrid DNA includes a mixture of 15 N and 14 N. The light DNA strands contain a pure 14 N. The diagram shows that only semi-conservative type of replication gave similar results conducted by Meselson and Stahl. Thus, both the conservative and dispersive modes of replication were disapproved.
Therefore, we can conclude that the type of replication in DNA is “Semi conservative”. The offsprings have a hybrid DNA containing a mixture of both template and newly synthesized DNA in the semi-conservative model. After each multiplication, the number of offspring will double, and half of the parental DNA will be conserved for the next generation.
What is the most challenging issue facing genome sequencing?
A. the inability to develop fast and accurate sequencing techniques
B. the ethics of using information from genomes at the individual level
C. the availability and stability of DNA
D. all of the above
Genomics can be used in agriculture to:
A. generate new hybrid strains
B. improve disease resistance
C. improve yield
D. all of the above
What kind of diseases are studied using genome-wide association studies?
A. viral diseases
B. single-gene inherited diseases
C. diseases caused by multiple genes
D. diseases caused by environmental factors
Successful DNA Plasmid Preps
Although DNA plasmid preps can return multiple forms of DNA, there is only one kind you want for successful cloning and transfection: supercoiled. Make sure you know how to increase your recovery of good quality supercoiled DNA. Did this help you understand why you get three bands when running plasmid DNA on agarose gels? Do you have any other plasmid prep tips? We’d love to hear in the comments.
Originally published October 8, 2014. Reviewed and republished April 2021.