PCR is a technique used to amplify specific regions of DNA. It requires DNA polymerase enzymes, primers that flank the target region, and cycles of heating and cooling. During each cycle, the target DNA is exponentially amplified, producing billions of copies that can be analyzed. PCR is used in forensic DNA profiling to amplify genetic markers found in crime scene DNA and a suspect's DNA, which can then be compared using gel electrophoresis to identify matching profiles.
PCR is a technique used to amplify specific regions of DNA. It requires DNA polymerase enzymes, primers that flank the target region, and cycles of heating and cooling. During each cycle, the target DNA is exponentially amplified, producing billions of copies that can be analyzed. PCR is used in forensic DNA profiling to amplify genetic markers found in crime scene DNA and a suspect's DNA, which can then be compared using gel electrophoresis to identify matching profiles.
PCR is a technique used to amplify specific regions of DNA. It requires DNA polymerase enzymes, primers that flank the target region, and cycles of heating and cooling. During each cycle, the target DNA is exponentially amplified, producing billions of copies that can be analyzed. PCR is used in forensic DNA profiling to amplify genetic markers found in crime scene DNA and a suspect's DNA, which can then be compared using gel electrophoresis to identify matching profiles.
PCR is a technique used to amplify specific regions of DNA. It requires DNA polymerase enzymes, primers that flank the target region, and cycles of heating and cooling. During each cycle, the target DNA is exponentially amplified, producing billions of copies that can be analyzed. PCR is used in forensic DNA profiling to amplify genetic markers found in crime scene DNA and a suspect's DNA, which can then be compared using gel electrophoresis to identify matching profiles.
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PCR & DNA Profiling
6C.4 & 6C.5
Polymerase Chain Reaction (PCR) • A laboratory technique used to amplify a specific target region of DNA. • E.g. a gene whose function a researcher wants to understand, or a genetic marker used by forensic scientists to match crime scene DNA with suspects. • The purpose is to make enough of the target DNA region that it can be analysed or used in some other way. Polymerase • Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. Taq Polymerase • High temperature is used repeatedly in PCR to separate the strands of the template DNA, causing DNA polymerase to become denatured. • The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus). • T. aquaticus lives in hot springs and hydrothermal vents ∴ its DNA polymerase is very heat-stable, with an optimum temperature of around 70°C. • This heat-stability makes Taq polymerase ideal for PCR. PCR Primers • Like other DNA polymerases, Taq polymerase can only make DNA if it's given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis. • In a PCR reaction, the experimenter determines the region of DNA that will be amplified by the primers. • PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length. • Two primers are used in each PCR reaction, and they are designed so that they flank the target region. • When the primers are bound to the template, they are extended by the Taq polymerase, and the region between them will be copied. The Steps of PCR • Denaturation (96 °C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step. • Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA. • Extension (72 °C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA. • This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2 - 4 hours, depending on the length of the DNA region being copied. • If the reaction is efficient, the target region can go from just one copy to billions. • It’s not just the original DNA that’s used as a template each time, the new DNA that’s made in one round can serve as a template in the next round of DNA synthesis. • The number of DNA molecules roughly doubles in each round of cycling. • Exponential growth Gel Electrophoresis • Used to visualise the results of PCR. • Gel electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an electric current, and it separates DNA fragments according to size. • A standard, or DNA ladder, is typically included so that the size of the fragments in the PCR sample can be determined. • https://learn.genetics.utah.edu/content/labs/gel/ • DNA fragments of the same length form a "band" on the gel, which can be seen by eye if the gel is stained with a DNA-binding dye. • For example, a PCR reaction producing a 400 base pair (bp) fragment would look like this on a gel: Applications of PCR • Using PCR, a DNA sequence can be amplified millions or billions of times, producing enough DNA copies to be analysed using other techniques • used for creating DNA profile for e.g. forensics or classification. • used to amplify genes associated with genetic disorders from the DNA of patients. • used to test for a bacterium or DNA virus in a patient's body: if the pathogen is present, it may be possible to amplify regions of its DNA from a blood or tissue sample. Sample problem: PCR in forensics • You have just received a DNA sample from a hair left at a crime scene, along with DNA samples from three possible suspects. • Your job is to examine a particular genetic marker and see whether any of the three suspects matches the hair DNA for this marker. • The marker comes in two versions. One contains a single repeat, while the other contains two copies of the repeat. • In a PCR reaction with primers that flank the repeat region, the first allele produces a 200 bp DNA fragment, while the second produces a 300 bp DNA fragment. • You perform PCR on the four DNA samples and visualize the results by gel electrophoresis, as shown. • Which suspect's DNA matches the DNA from the crime scene at this marker? Introns and Exons • Functions? • Which does DNA Profiling use? • Micro-satellites: 2-6 bases repeated 5 to 100 times • Mini-satellites: 10-100 bases repeated 50 to several hundred times • Number of repeats linked to relatedness • 11+ matching micro-satellites = same individual