2. molecular diagnostics corrected

what are the basic components of polymerase chain reaction (PCR)

• primers

• template DNA

• dNTP

• taq polymerase (thermus aquatics)

what are dNTPs

the A, C, T, G

what are some important concepts of PCR

• there are 4 bases (dNTPs)

• each of the bases can only pair up with its complementary base (A ↔ T/U, C ↔ G)

• a primer is a short stretch of synthetic DNA (oligonucleotide) that can pair up with the front part of DNA to initiate DNA polymerization

what can the DNA polymerase do

DNA polymerase can copy the template DNA

what are the different steps to PCR and their temperatures

1. denaturation at 95°C : getting the template DNA denatured

2. annealing at 55°C - 60°C : the primers bind to the template

3. extension at 72°C : the Taq polymerase extends primers

why is good primer design essential for PCR

because the primers determine which location is amplified

what is the result if the primer melting temperature is too high or too low

too low:

• poor hybridization and unstable binding → weak amplification and thus has no/faint bands

• hence primers fail to anneal effectively at higher temperatures, thus having low yield

too high:

• non-specific binding and sticky primers → non-specific bands and primer dimers

• hence primers anneal to similar, non-complementary regions with multiple products and background noise

what is the effect of the primer melting temperature being just nice

• specific binding and efficient hybridization → strong, specific band

• hence it is efficient, specific amplification with high product yield and a minimal background

what are the components of a PCR machine

• heated lid

• 24-well thermal block

• optical system

• LCD

• keypad

• LEDs

what is real-time PCR or quantitative PCR (qPCR)

• when the SYBR green fluorescence increases up to 1,000-fold upon intercalation with double stranded DNA

• add this dye into the PCR reaction, and monitor at every cycle

when can qPCR results be comparable

• when they are performed on the same machine settings with the same reagents

how do people divide samples into 10,000 1nL partitions

microfluidics