PCR Flashcards
What is PCR
Polymerase Chain Reaction is a method that is used to amplify a specific DNA segment of up to 6kb. It is faster and more convenient than cloning.
Explain how Polymerase Chain Reaction works to amplify a DNA segment
A heat denatured DNA sample is incubated with DNA polymerase, dNTPs and 2 oligonucleotide primers. The primers flank(occurs on either side) the DNA segment to direct DNA polymerase to synthesize new complementary strands. Multiple cycles of this process, each doubles the amount of DNA, amplifies the DNA greatly. Each cycle involves denaturing the DNA strands by increasing the temperature to 95 degrees, the temperature is then lowered to allow the primers to anneal to their complementary segments on the DNA and the DNA polymerase synthesizes new strands.
In PCR the enzyme DNA polymerase can perform many cycles, how is this possible
Heat-stable DNA polymerase is used, eg. Taq DNA polymerase or Pfu DNA polymerase, so a new enzyme is not needed after each denaturation
What does PCR require in order to carry out many amplifications
As long as there are sufficient quantities of dNTPs and primers the PCR is carried out simply by varying the temperature after each cycle in an automatic device called a thermocycler.
Explain the exonuclease activity of DNA polymerase I
DNA polymerase I has polymerase activity, as well as 2 hydrolytic activities: 5’-3’ exonuclease activity and 3’-5’ exonuclease activity. Taq DNA polymerase I lacks 3’-5’ exonuclease activity, but Pfu DNA polymerase I does contain it.
Theoretically how much does the DNA sequence increase by in 20 cycles
2^20 or 10^6 fold with high specificity
In reality how much of the DNA sequence is yielded after 20 cycles
The number of sequences doubles after each cycle until there is more primer-template complex that accumulates than the DNA polymerase can extended during a cycle, so about 20% of the theoretical yield
PCR has been shown to amplify a target DNA present only once in a sample of 10^5 cells, what does this demonstrate
PCR can occur without the purification of DNA
What is the clinical use of PCR
It is used for the rapid diagnosis of infectious diseases and the detection of rare pathological events such as mutations that lead to cancer
What is the forensic use of PCR
The DNA from a hair, blood or sperm can be used to identify the donor. This is most commonly done through the analysis of STRs(Short Tandem Repeats). STRs are segments of DNA that contain repeating sequences of 2-7 bp that are scattered throughout the human genome. The number of tandem repeats(n) in STRs vary , therefore they are markers of individuality.
How can STRs be PCR-amplified
Primers flank the STR and anneal to the complementary strands
How can we measure the number of repeats in that STR from an individual
The measurement of its molecular mass through acrylamide gel electrophoresis or by direct sequencing
How can PCR be used to prove or disprove familial relationships
Only the tips of the Y chromosomes undergo recombination with the X chromosome and the rest of the Y chromosomes remains untouched. When the STR-based haplotypes are identical in males it proves that they are related
How is it possible to use PCR to amplify RNA
By reverse transcribing it into a complementary strand of DNA(cDNA) through the action of an enzyme called RNA-directed DNA polymerase( also called reverse transcriptase). This enzyme which is produced by retroviruses uses an RNA template, otherwise it is similar to DNA polymerase I.
What is asymmetric PCR
A method used to rapidly generate single-stranded DNA. A small amount of one primer is used so it gets exhausted after several PCR cycles. In the rest of the cycles the strand that contains the primer that is in excess is synthesized.
