Lectures 7+8: Sequencing and primer design

What are the three stages of a PCR reaction?

Denaturation, annealing, extension

At what temperature does denaturation take place?

92-95 degrees

At what temperature does annealing take place?

55 degrees

At what temperature does extension take place?

72 degrees

At what pH does a PCR take place?

9

Why are GC rich sequences difficult to amplify?

What are the problems with PCR? [5]

Contamination, inappropriate priming, taq errors, oprimisation, difficult sequences

What is site-directed mutagenesis

Introducing changes into PCR products by using slightly modified primers

What is the other name for dideoxy method of sequencing?

Sanger sequencing

What is the Gilbert-Maxam method of DNA sequencing?

An outdated method based (from 1977) based on chemical modification and cleavage of DNA

What principle does Sanger sequencing work on?

Replication of a DNA sequence in vitro using a DNA polymerase, dNTPs and dideoxy chain terminators (ddNTPs)

What part of the DNA molecule provides the energy to join it onto a growing chain of DNA?

The triphosphate

How is a ddNTP structurally different from a dNTP?

Missing the OH group on the 3’ end

What are the differences between manual and automated sanger sequences?

• Manual - typically uses large flat acrylamide gels, radioactivity and x-ray film to separate and detect products

• Automated - uses liquid capillary gel machines and fluorescent tags to separate and detect products

How is template DNA generated?

Millions of copies are generated using plasmid cloning or PCR

How many bases usually are there in a primer for a sequencing reaction?

17+ bases because it is crucial that the reaction starts in the same place

How are completely unknown sections of the genome sequenced (ie. in situations where a primer cannot be used)

Cloning the unknown piece of DNA into a plasmid vector and getting a continuous read around the vector

Use a primer that binds to the plasmid near the cloning site

What is liquid gel chromatography?

An automated method for reading DNA sequences

DNA travels through the gel in the tube (smaller fragments travel more slowly)

Each ddNTP is labelled a different colour and fluoresce as they pass a laser

A detector is wired up to a computer opposite the laser

How many parallel capillaries are used in liquid gel chromatography?

96

What structural feature differentiates ribose from deoxyribose?

Ribose has a hydroxyl group at the 2nd Carbon but deoxyribose just has an H

What are the major features that are considerations of primer design

• Avoiding primer dimers and hairpins

• Annealing temperature (increased by length and GC content)

• Avoiding common repeat sequences

What is the difference between a microsatellite and a minisatellite?

Microsatellites 2-6bp

Minisatellites 50-100bp

Why are minisatellites used in forensic identification

Highly variable numbers

What are line elements/ sine elements? How do they relate to primer design?

DNA with ‘selfish’ properties which causes the same sequence to be found in multiple places in the genome.

When designing primers they are run against a database of known DNA to make sure it’s not one of those sequences because this would interfere with PCR if the wrong regions are amplified

What is a GC clamp?

At least one G or C in the last five bases of a primer