L4 Analysing DNA

Question 1

Verifying correct DNA is cloned

Answer 1

If the nucleotide sequence of the cloned fragment is known, restriction analysis.

A recombinant plasmid is mixed with non-recombinant, the colonies are grown and cultured in solution. > Gel Electrophoresis.

Restrictions:
-complement analysis with flanking the cloning site and cloning sites internal to the cloned sequence.

Question 2

Analysis of high number of clones - DNA hybridisation

Answer 2

Nucleic acid hybridisation method.
Needs to have background information on DNA i.e nucleotides
Based on the complementary base pairing of nucleic acids. reverse of dna denaturing.

It is then labelled with T4 polynucleotide kinase and radioactive ATP.

This molecule is used as a PROBE

Question 3

Southern blot

Answer 3

1) Gel electrophoresis
2)DNA frags are blotted into a membrane
3)DNA is “cross linked” (binded)to membrane by heat or UV
4)Probe is added to membrane via H-bonds
and undergoes complementary base pairing.
5)Excess probe is washed away and membrane is analysed by X-ray to detect position.

Question 4

How to screen numerous clones using DNA hybridisation

Answer 4

1) Use a nitrocellulose membrane to extract colonies from petri dish.
2) Lyse bacteria and denature DNA (exposure)
3) Incubate with probe and wash
4) Align membrane with petri dish to determine which colonies are replica.

Question 5

DNA sequencing (post cloning)

Answer 5

Based on DNA strands complementary and DNA replication mechanism.

Question 6

DNA replication overview

Answer 6

Semiconservative method - parental strands serve as templates

PRIMER- used to located beginning of replication at 3’ end.

DNA polymerase adds nucletides from 5’ to 3’

Each nucleotide added releases pyrophosphate (PPi)

Leading enzyme complex elongates

Lagging enzyme complex creates new fragments

Question 7

DNA synthesis proof reading - Exonuclease

Answer 7

Works from 3’ to 5’ direction

Question 8

DNA sequencing - Sanger method (chain termination)

Answer 8

Based on DNA replication and PRIMER.
primer binding sequence must be known.

Then ddNTP is incorporated to growing strand, preventing DNA synthesis.

The primer is radioactively labelled.

A mixture of individual nucleotides, DNA polymerase and ddNTP is then added to DNA strand.

Gel electrophoresis is then used to determine which base was incorporated.

This can also be automated by using fluorescently labelled nucleotides with each ddNTP labelled with different dyes