Ch 9.3 & 9.4 - Molecular Genetic Analysis

polymerase chain reaction (PCR)

• DNA synthesis in a test tube

• used to amplify a target sequence in order to study it

components of a PCR

• template DNA (from genomic extraction)

• Taq polymerase (thermal stable DMA polymerase)

• dNTPs (building blocks of DNA)

• primers (oligio) (short sequences that define the outer bonds of a target sequence, provide free 3’OH for Taq, used in pair, forward and reverse)

• buffers

What do the dNTPS and primers give a chance for?

chance to incorporate and label other sequences

the cycles/stages of PCR

1. 95 degrees: denature- hydrogen bonds are broken and ssDNA (single stranded DNA) is formed

2. 55-65 degrees: annealing- primers anneal to target sequence

3. 72 degrees: elongation - taq polymerase synthesizes new DNA

What type of amplification is seen in PCRs?

exponential amplification (2ⁿ)

Dideoxy-DNA (Sanger) sequencing

• similar to PCR but chemically different

• relies on using a terminator nucleotide-dideoxyribonucleotide triphosphate (ddNTP)

  • this is used at lower molar ratios than dNTPs

• full synthesis will occur but will sometimes incorporate ddNTPs and terminate

What is the difference between dNTPs and ddNTPs?

ddNTPs lack a 3’OH

components of dideoxy-DNA sequencing

• template DNA

• Taq polymerase

• dNTPs and ddNTPs

• primers (only one primer used for each reaction for linear amplification NOT exponential)

• buffers

How were dideoxy-DNA sequencing historical done?

• in 4 separate test tubes, each with a different labeled ddNTP

• during DNA synthesis, some ddNTP gets incorporated

• at end there are fragments differing by one nucleotide each, ending with its respective ddNTP

• all 4 samples are run side by side

• the sequence is read up the gel across all 4 lanes

How are dideoxy-DNA sequences done today?

• all 4 ddNTPs are used at the same time in the same reaction tube

• each ddNTP is labeled with a different fluorescence color