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LAB 10 WHAT IS PCR AND HOW DOES IT WORK? (10 points) EXPERIMENT OBJECTIVE: The objective of this experiment is for students to gain hands-on experience in the principles and practice of Polymerase Chain Reaction (PCR). Reaction (PCR) has had an extraordinary impact on various aspects of biotechnology. PCR has revolutionized research and diagnostics bi based molecular ology PCR is a simple, accurate and highly reproducible procedure. The technology introduced an important advantage to molecular biology. It provides the ability to start with a small amount of DNA and to be able to amplify it so that there will be a sufficient amount of DNA to perform experiments. It is analogous to a radio or stereo amplifier where radio wave signals which are normally not heard are amplified so we can hear music. Since the first application of PCR to detect sickle cell anemia, a large number of diagnostic tests have been developed and are becoming routine tests. PCR is also used in genome projects for DNA mapping and sequencing and-is being applied to forensics, patermity determinations, as well as the determination of evolutionary relationships. In all these cases the DNA samples that are extracted are limited and PCR amplifies segments of DNA that become the subject for further analysis and study. In a PCR reaction, the first step is the preparation of the DNA sample that is extracted from tissues or various biological sources. In PCR experiments, the DNA or gene to be amplified is referred to as the target and the synthetic oligonucleotides used are referred to as primers. A set of two primers (a forward and reverse primer) usually ranging between 20 and 45 nucleotides are chemically synthesized to correspond to the two ends of the gene to be amplified. Each primer binds to one of the two DNA strands and is the initiation point of the amplification. The primer concentrations are always in excess of the target gene to make possible subsequent priming. The exact nucleotide primer sequences for a specific amplification reaction are determined to yield the best conditions (hybridization) for template-primer formation. The specificity of DNA synthesis is dictated by the Watson – Crick base pairing roles and is directed by the template DNA. The strand being syuthesized is complementary and antiparallel to he templaie DNA strand. De novo DNA synthes catalyzed by DNA polymerase cannot occur without a primer having a free 3′ terminal hydroxyl group, which is required for the addition of the next nucleotide. The primer is antiparallel and is base paired to the template strand. An overview of the PCR reaction is shown on the next page A typical PCR reaction mixture contains DNA, Tay DNA polymerase, and the four deoxynucleotide triphosphates in the appropriate buffer. The incubation mixture is then exposed to three step temperature cycle which is repeated. The first temperature is 94°C to melt the hydrogen bonds between the two strands of DNA. The temperature is then dropped to between 42° and 60 C idize the two primers on the two DNA target strands. The temperature is then increased to 72°C, which is the optimum temperature for Tag DNA polymerase. At this temperature, the DNA synthesizes the opposite strand of DNA using the original strands as templates. These temperature cycles are repeated 20 to several hundred times. This process is made efficient by placing the reaction tubes in specifically designed thermal cyclers which are programmed to alternate temperatures rapidly and accurately. The amplified product is then detected by separating 73 Show transcribed image text
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