PCR

DNA helices

formed vt DNA because of base pairing and base stacking (ring stacking)

Base stacking

conjugated ring systems are flat and hydrophobic favoring stacking interactions

affected by hydrophobic effects, Van Der Waals interactions, electrostatic interactions —> base stacking

not base specific

Base pairing and base stacking

combination with weak interactions holds double helices together

Why is it good for a nucleic acid to be held together by many weak interactions?

DNA needs to be separated frequently for replication and transcription

PCR

polymerase chain reaction, amplify specific DNA sequence that you choose

Why do PCR?

• diagnostic test: covid, mutations, paternity/maternity

• Identify biological matter more broadly (find specific gene etc)

• Make DNA and analyze

• measure gene expression

• recombinant DNA tech

Denature

separating and melting

Annealing

coming together

Tm

melting temperature, about half of DNA molecules denatured or annealed

PCR step 1

heat to separate strands: denaturation or melting

PCR step 2

add synthetic oligo nucleotide primers; cool

primers anneal to edgue of gene we want to amplify

Oligo

short strand; aka primers or oligos

PCR step 3

add thermostable DNA polymerase to catalyze 5’ —> 3’ DNA synthesis

add nucleotides to 3’ end more DNA

One cycle

1st 3 steps of PCR

DNA polymerase

always adds nucleotides to 3’ end of primer

Denature temperature

95 deg C

Annealing temperature

50-65 deg C depends on primers

Extension/elongation temperature

72 deg C

More G-C rich primer or longer primer

higher annealing temp

Factor DNA fragment amplified

2^(# of cycles)

Things needed for PCR

1. template DNA

2. specific primers

3. thermostable DNA polymerase '

4. dNTPs

5. appropriate buffer

Gel electrophoresis

determine size of DNA molecules

smaller molecules move more quickly

DNA/RNA negative so pulled to anode (+)

Forensic analysis

PCR to amplify variable repeat sequences