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PCR Electrophoresis

PCR Electrophoresis. By C. Kohn, Agricultural Sciences, Waterford WI. Genetic Testing. The Sanger Method enables scientists to read DNA letter by letter. This test made the Human Genome Project, and the sequencing of many other species, possible.

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PCR Electrophoresis

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  1. PCR Electrophoresis By C. Kohn, Agricultural Sciences, Waterford WI

  2. Genetic Testing • The Sanger Method enables scientists to read DNA letter by letter. • This test made the Human Genome Project, and the sequencing of many other species, possible. • The problem with the Sanger Method is that it is slow and expensive. • It wouldn’t work to use it for day-to-day needs. • The Sanger Method provides scientists with every possible letter in a genome. • This amount of information is not always necessary. Source: en.wikipedia.org

  3. DNA Fingerprinting • When we simply need to identify the sourceof DNA, or a few genes, we can use simpler and less-expensive tests. • Often we don’t need to know the entire genome of person – we just need to know if their DNA is a match to another sample of DNA, or if their DNA contains a specific set of genes. • To determine this, we would create a DNA Fingerprint. Source: buzzle.com

  4. Human DNA • DNA fingerprinting works for any kind of DNA, even human DNA. • This is important because 99.9% of human DNA is the same. • This means that you have 99.9% of the same DNA as the person sitting next to you. • The 0.1% difference between two peoples’ DNA means that 3 million bases are different – this is more than enough to allow us to pinpoint the source of DNA down to a specific individual. • DNA Fingerprinting is when a geneticist examines the 3 million base pairs that could be different for any variations that are unique to an individual. Source: dnafingerprintingx.yolasite.com

  5. Creating a DNA Fingerprint • To create a DNA fingerprint, we have four steps: • 1. Collect a sample of DNA • 2. Amplify specific variable regions of the DNA • 3. Count repeated sequences (STRs) • 4. Look for matches from collected samples to determine what samples are from the same sources • For example, if you found DNA at the scene of a crime, you could collect DNA from a suspect and see if it matches the DNA found at the crime scene. Source: dna-rna.net

  6. Collection & Amplification • The first step in solving this hypothetical crime would be to collect a pure, untainted sample of DNA. • We can’t have multiple sources of DNA in the same sample. • Next, scientists have to amplify regions of the DNA that have a lot of variety from person to person. • We can’t look at regions of DNA that are similar from person to person. • Typically, exonsare going to be very similar from person to person. • Vice versa, introns (sections of DNA that do not code for proteins) have a lot more variability and work better for DNA fingerprinting. • Introns tend to have small sections that repeat over and over. Source: simple.wikipedia.org

  7. STR’s • When scientists compare the intron sampling regions of the DNA, they are comparing Short Tandem Repeatsof DNA, or STRs. • An STR is a short unit of DNA (4-5 bases long) that repeats multiple times in the DNA. • For example, you might a “AATG” section of DNA that repeats over and over  ATCT.AATG.AATG.AATG.AATG.CAG…” • In this case, there would be 4 STR repeats of AATG. Source: nfstc.org

  8. Importance of STR’s • STRs are critical because they represent the areas of greatest variability from person to person. • While the DNA that codes for functional proteins will be very similar from person to person, the STRs in introns are the areas of greatest variety and are the only way to create a unique DNA fingerprint for each person. • While you may have the same number of STRs at one particular region in your DNA as someone else, the likelihood of having the same number of STR repeats at every region in your DNA and someone else’s DNA is next to impossible. Source: bcs.whfreeman.com

  9. Example • For example, if you look in the chart below, you can see 5 regions where STRs were measured and counted. • In the first column, you can see the number of repeats at each STR location. • For each suspect, you can see if their number of STR repeats at each location matches. • In this case, the second suspect’s DNA is a match to the crime scene.

  10. How we do it • So how can we tell how many STRs each person has at each sampling site? How can we determine a person’s or organism’s DNA fingerprint? • The key to determining this information is a test called Polymerase Chain Reaction, or PCR. • PCR is sort of like a molecular copy machine. • It can quickly, easily, and cheaply make many copies of a specific section of DNA. • It does not work to read all of an individual’s genome, but it does work well for small sections of DNA. Source: scq.ubc.ca

  11. PCR works first by separating the dual-strand of DNA into two individual strands using heat. • Each side can then be copied by polymerase enzymes, creating two strands from one. • This process is repeated over and over (2 becomes 4, 4 becomes 8, 8 becomes 16…...) • It can amplify a specific sequence of DNA billions of times! PCR Animations 1, 2, 3, 4, 5

  12. Taq Polymerase • To denature the DNA, or separate the double strand into two single strands, we have to heat the DNA. • One problem with this method was that the heat also destroyed a normal polymerase enzyme. • To make this technique work, scientists use the Taq polymerase. • Taq polymerase is found in bacteria that live in the hottest environments on earth (such as the inside of the geysers at Yellowstone where it was first discovered). • The Taq polymerase is able to function in the hot/cold/hot kind of conditions used for PCR.

  13. After amplification by Taq polymerase, each DNA sample is cut up into fragments by restriction enzymes (chemical scissors) • Remember that restriction enzymes recognize and cut within specific DNA sequences. • These specific DNA sequences are located at different places along the strand of DNA, depending on the source. • Because of this, each DNA sample will have a unique banding pattern. DNA Seq. 1 DNA Seq. 2 The same restriction enzyme will cut the same gene from different people in different ways.Chunks of DNA will be larger if they have longer STR repeats.

  14. Restriction Fragments • For example, EcoRI is a restriction fragment that always looks for the GAATTC sequence. • It will always cut DNA between the G and the first A. • This sequence is likely to show up multiple times in a normal DNA segment. • Because this segment will occur different places in different DNA, it will create uniquely sized ‘chunks’ of DNA for each person depending on the number of STRs.

  15. Gel Electrophoresis • Once we’ve made billions of copies of the same section of DNA and have cut them with a restriction enzyme, we have to run the DNA through a gel. • This is necessary in order for scientists to determine how much DNA was in that particular section that was copied. • If we know how much DNA was in that section, we knowhow many STR repeats werein that section. • Shorter fragments of DNA will travel further than longer chunks. • Known quantities of DNA providea basis for comparison in the gel.

  16. Gel Electrophoresis • A few drops of each amplified and cut DNA sample are placed in a small pocket, or well, at one end of the gel. • The other end of the gel has a positive charge. • All DNA molecules are negatively charged, so they move through pores in the gel toward the positive pole. • The phosphate in the DNA gives it a negative charge.

  17. Gel Electrophoresis • The shorter DNA fragments slip more easily through the pores of the gel than the longer fragments. • I.e. it would be easier for you to swim to the other side of the pool if you were wearing a Speedo than if you were wearing baggy shorts. • After a set period of time, the shorter DNA fragments will travel farther through the gel. • The shorter fragments will be closer to the positive end of the gel than the longer fragments.

  18. Treating/Reading the Samples • In the last step, the gel is treated with a stain that makes the DNA visible under ultraviolet light. • The DNA fragments show up as a series of bands in each "lane" of the gel. Each band consists of many DNA fragments of one particular size. • That size is determined by the number of STR repeats. • The pattern of bands will be different for each sample because each sample has a unique set of DNA fragment lengths.

  19. Genetic markers • A genetic marker is a gene that is found at a known location and produces a predictable pattern. • When DNA fingerprints are used in court as evidence, genetic markers from these highly variable noncoding regions are used. • Genetic markers can occur in alleles for diseases or other traits. • We can compare the banding patterns of a patientto known patterns from people with and without thedisease to see if they match one or the other. • If the patterns of the patient match the markers from the known samples with the disease, we know that that patient would also have that genetic disease • Whenever we analyze the banding patterns from PCR/Electrophoresis, this is known as Restriction Fragment Analysis.

  20. Summary • DNA sections are amplified tens of billions of times by Taq polymerase in the process of Polymerase Chain Reaction, or PCR. • The amplified DNA is cut into chunks using a restriction enzyme. • The cut pieces of DNA are put into wells in a gel and pulled through the gel using electricity in a process called Gel Electrophoresis • As the fragments move through the gel, they create a unique banding pattern. This is due to the fact that the larger pieces of DNA move more slowly than the small pieces. • Larger chunks have more repeating sections, or STRs, than smaller chunks. • The unique banding pattern (Restriction Fragment Analysis) enables scientists to determine the source of a sample of DNA by comparing it to collected samples.

  21. Extra Resources • Gel Electrophoresis Virtual Lab • DNA Detective; Who Dun It ? • Polymerase Chain Reaction Quiz 1, 2, 3 • DNA Fingerprinting Tutorial & Quiz (advanced) • DNA Fingerprint Quiz 1, 2 ,3 , 4, 5

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