Question

DI Succuleluujective vi, a 16 points) PCR is a powerful tool used to isolate and make large quantities of a defined DNA fragment from a complex genome. PCR takes advantage of electrophoresis and synthetic oligonucleotides. Its starting material can be as small as a single cell. Because amplification is exponential, a single DNA molecule can be copied into hundreds of copies in a single day. Once a reference DNA sequence has been established, PCR can be used to pick out variant forms of that sequence in any DNA sample. PCR amplifies any sequence between the ddNTPs used even if the order of bases is slightly different. CT GA Sanger Sequencing (Objective vi; 3 min) (6 points) You've been tasked with finding an autoradiogram of a DNA sequencing gel that's been lost. After a day of searching, you decide it would be easier to recreate the data. The DNA sequence you're looking for is: 5'-ACTATCATTTGGATGCT-3' and the sequencing primer was 5'-CAAATG-3'. Reconstruct the gel in the box to the right.
find three errors in the pragraph and reconstruct the gel

Answer 1

Errors

PCR does not isolate DNA molecules. It simply amplifies the template DNA.

PCR does not copy a DNA molecule. It simply amplifies a template DNA several folds.

PCR amplifies the template DNA between the primers (or even outwards from the primers in case of inverse PCR) and not between the ddNTPs.

Sanger sequencing

5' ACTATCATTTCGGATGCT 3'

3' GTAAAC 5'

CT GA GTAAACCTACGA GTAAACCTAOG GTAAACCTAC GTAAACCTA GTAAACCT GTAAACC

Sanger sequencing involves the use of ddnTPs (lack OH at 3' carbon of ribose sugar) which cannot be extended by DNA polymerase.

The method involves chain termination by incorporation of ddNTP and the then separation on a gel.

A mixture of dNTPs and is used in 4 separate reaction wells each containing only 1 type of ddNTP.

A primer (radiolabelled) is annealed to the template DNA which would be extended by the polymerase.

The mixture of DNA fragments produced after the reaction is complete, in each well is separated on a Polyacrylamide gel which can resolve fragments with differences in 1 base.

The bands can be visulaiized via autoradiography.