L13- Recombinant DNA Tech 2

PCR

• amplifies specific target DNA from very small quantities

• Copies a specific DNA sequence through in vitro rxns

Requirements for PCR

• Template DNA

• Polymerase + buffer (Mg+)

• Primers

  • define the specific region we are copying

• Nucleotides (dNTPs)

• Thermocycler

Original heat stable enzymes were isolated from

• hot springs bacterium= Taq pol

• Don’t denature at high temperatures

Steps of PCR

• Will create nearly unlimited amts of DNA from a template

1. Denaturation 45-65 °C

• Separates DNA

2. Annealing 45-65°C

• Allow primer to bind (anneal) before strands come together

• This primer is complementary to target region

3. Extension ~72 °C

• Taq pol synthesizes DNA 5’-3’

1x→2x→ 4x→ 8x (now have target DNA by itself) after 3 rounds

How to calculate copies of DNA created in PCR rxn

• 2^n copies

  • n= # PCR cycles

• For target copies of DNA 2^n- n

PCR primers

• Determine region to be amplified

• Provide starting point for Taq pol

• SS DNA

• Complementary to target DNA

• aprox 20 nt long

Annealing temp calculations

• Annealing= Tm- 5°C

• Tm= 81.5 +0.41(%GC)-(675/N)

  • Use whole number for %GC not decimal

  • N= total # nucleotides

• DONT USE THIS but know: 2(A+T) + 4(G+C)→ undereps Tm

Benefits of PCR

• More sensitive and faster than cloning

• BUT there are limiations

Limitations of PCR

1. Specific primers require that some sequence information be known

• if find new bac species cant do PCR bc need to make complementary primer

2. Cannot amplifiy very long segments of DNA

• Cannot do genome/entire chrom w/out 1st breaking into pieces OR find some other enzyme not Taq that synth longer pieces → more

3. Taq polymerase doesn’t proofread

• If seq needs to be abs. perfect there might be some error that you gotta check. BUT there’s other bac. enzymes that proofread→

4. The sensitivity can result in amplification of contaminating sequences

• Ensure only template in tube is what we want or else get PCR of something else→ detective DNA in suspect sample

What’s Reverse transcription PCR (RT-PCR), what are its uses

• PCR from mRNA (template)

  • or any other RNA but mRNA most common

• mRNA→ cDNA

• cDNA amplified by PCR

Uses

• Testing for presence of OR amplfiying an RNA

  • mRNA→ reflects which genes are active, compare expression bw diff cell types like kidney vs other tissues. Compare normal vs diseased cells.

  • RNA viruses→ test for presence covid/HIV

qPCR/ qRT-PCR allows for

• accurate quantification of mRNA levels

• Includes RT first (RNA→ cDNA)

How it works

• PCR (amplification) occurs in presence of fluorescnet markers (e.g SYBR green)

• Fluorescence increases as more DNA is produced each cycle

  • As PCR produces more double-stranded DNA → more dye binds → more fluorescence

• The signal is measured in real time + compared across cycles

Site specific mutagenesis of DNA

• Using mutagens to gen mutations often leads to multiple non specific mutations

• But this method allows for directed mutagen

  • Can mimic human mutations in other organisms

  • “humanization”