41 - Recombinant DNA

Reverse transcriptase

Synthesize DNA from RNA, used by viruses

What does gel electrophoresis mostly sort/separate by?

Length

PCR

Amplifies desired region of DNA between 2 primers

3 Steps of PCR

1. Denaturation by heating

2. Annealing primers to ssDNA

3. Extension by thermostable DNA polymerase and dNTPs

What are the 4 things needed (besides DNA template) for PCR amplification?

dNTPs, Buffer (Mg2+), primers, and DNA polymerase (Taq or Pfu since it must be thermostable)

RT-PCR

Amplification of RNA instead of DNA using reverse transcriptase, can be used to detect COVID-19

Dideoxynucleotides: What are they and what is their function in gel electrophoresis?

Nucleotides that don’t have an OH on the 3’ sugar, they terminate chain growth

Do smaller or larger fragments travel further in gel electrophoresis?

Smaller fragments travel further, so the bottom band is the first nucleotide at the 5’ end

What are the old and new methods of sequencing DNA?

Old - automatic sequencer

new - high throughput sequencing (pyrosequencing)

Synthesized strand:

5’ TTCAGAAGT 3’

3’ AAGTCTTCA5’

Thus

5’ ACTTCTGAA 3’