BIO615 CH 4.1 PCR

Reorder the following principles of PCR.

• Annealing (~55)

  - Cool to allow primers to form hydrogen bonds with ends of target sequence.

• Extension (~72)

  DNA polymerase adds nucleotides to the 3’ end of each primer.

• Denaturation (~95)

  - Heat briefly to separate DNA strands. Break hydrogen bond.

What is the definition of PCR ?

• A method to amplify DNA, in which small amount of DNA can be copied in large quantities over a short period of time

• A method to detect the genetic materials of DNA in small quantities.

Match the following solution for PCR trouble shooting.

• Tm = 4(G+C) + 2(A+T)

• Primer Melting Temperature (Tm)

• Lower annealing temperature

• Unspecific/Numerous band

• Increase annealing temperature

• No bands

Reorder the following steps of PCR

• Raise the temperature to ~72°C —> allows the thermophilic polymerase to add complementary nucleotides to the 3' end of the primers

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• Then heat the mixture to ~95°C, melting (denaturing) the DNA in mix & separating the strands

• Place PCR tubes into a thermal cycler

• The mix is then cooled to ~55°C —> allows primers to bind 3' ends of both target DNA strands

What determines the Primer Melting Temperature (Tm) ?

• primer length

• base composition

• concentration

• salt concentration

• initial temperature of the mix

What is RT-PCR ?

• Technique to convert RNA into DNA and amplify it using PCR.

• Technique used to measure the amount of DNA during PCR in real-time

What is qPCR ?

• Technique to convert RNA into DNA and amplify it using PCR.

• Technique used to measure the amount of DNA during PCR in real-time

Why Use qPCR?

• Quantifies DNA: Measures the exact amount of DNA at any cycle, which helps in gene expression studies or detecting low amounts of DNA (e.g., in viral load testing).

• More Sensitive: It’s much more sensitive than conventional PCR because it provides real-time data rather than just a final result.

Reorder the following steps in RT-PCR

• Convert mRNA -> cDNA

• Use forward primer turn cDNA -> dsDNA

• Use reverse primer

• Starts with mRNA

• PCR

Reorder the following

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