Analysis of Nucleic Acids

Question 1

Describe the creation of recombinant DNA molecules in In vivo cell-based cloning

Answer 1

Target DNA and a Replicon cut with the same restriction endonuclease. Mixed and then DNA ligase added

Question 2

What is a replicon?

Answer 2

Sequence capable of independent replication

Question 3

Name 3 Replicons

Answer 3

Plasmin, Lambda phage, Yeast artificial chromosome

Question 4

What is done with Recombinant DNA in cell-based cloning?

Answer 4

Transformed into a host cell e.g. Yeast/bacteria, which will replicate the replicon and itself

Question 5

How is a host cell containing recombinant DNA selected and replicated in cell-based cloning?

Answer 5

The host cell is placed on a agar plate with an antibiotic, so only the cells containing target DNA and Antimicrobial resistance gene can survive.
Colonies transfered to growth medium to replicate

Question 6

Why is the same restriction endonuclease for both target DNA and the replicon in cell-based cloning?

Answer 6

So the two nucleotides are compatible with each other

Question 7

What is a restriction endonuclease?

Answer 7

Enzyme that cleaves DNA at specific 4-8bp palindromic sequence to introduce blunt/sticky ends

Question 8

What does gel electrophoresis do?

Answer 8

Separates DNA fragments based on size

Question 9

How does gel electrophoresis work?

Answer 9

DNA is negatively charged due to phosphate backbone.

When fragments placed in solution and current is applied, they travel towards positive electrode

Question 10

Which DNA fragments travel further in gel electrophoresis?

Answer 10

Smaller fragments as they are less hindered by mass

Question 11

What is Nucleic acid hybridisation?

Answer 11

Method of detection of specific sequences using labelled probes

Question 12

How can fragments from electrophoresis be identified?

Answer 12

Transfer to hybridisation assay

Question 13

What is a hybridisation assay

Answer 13

Target DNA immobilised on solid support which readily binds single stranded nucleic acids, then probe DNA added in solution over membrane. Denaturing then allows the probe dsDNA to become ssDNA, then allowing the probe to bind to any target DNA if it is present, and any excess washed away

Question 14

How can probes be detected?

Answer 14

Can be radioactively or fluorescently labelled

Question 15

What are the targets and probes in:
Northern Blot
Southern Blot

Answer 15

Northern: RNA target + DNA probe
Southern: DNA target + DNA probe

Question 16

What are the targets and probes in the following assays:
Colony Blot
Tissue in situ

Answer 16

Colony: Bacterial DNA target + DNA probe
Tissue: RNA target + RNA probe

Question 17

What are the targets and probes in:
Chromosome in situ
Reverse hybridisation (microarray)

Answer 17

Chromosome: Chromosome target + DNA probe
Microarray: immobilised DNA or oligonucleotide probe + target DNA solution

Question 18

How would you perform southern blotting ?

After electrophoresis carried out

Answer 18

Fragments immobilised on nylon filter and radio probes added to bind to comp DNA. X-ray film used to detect probe position

Question 19

What is the melting temperature of DNA at 40% GC?

Answer 19

87 oC

Question 20

Why might the Tm of DNA be higher?

Answer 20

If it contained higher % GC, as more H bonds to break

Question 21

What is hybridisation stringency?

Answer 21

The power to distinguish between related sequences

Question 22

What happens at high hybridisation stringency?

Answer 22

Duplexes only form between perfectly complimentary strands

Question 23

How might lower hybridisation stringency be encouraged?

Answer 23

Lower temp, higher Na+ conc

Question 24

What is required for PCR?

Answer 24

2 Primers
Target DNA
Taq polymerase
dNTP's
(Heat)

Question 25

How is PCR carried out?

Answer 25

1. DNA denatured at 95oC
2. Annealing at 50-60oC with dNTPs by Taq poly
3. extension at 72oC
4. Repeat many times in thermocycler

Question 26

How long is a PCR primer and why?

Answer 26

~20bp

Long enough to be specific to target.

Question 27

How is a PCR primer designed?

Answer 27

Avoiding tandem repeats to prevent hairpin formation.
%GC standardised to give roughly equal Tm for each primer.
Complementarity of 3’ ends avoided to prevent primer dimerisation.

Question 28

Why are 2 primers required for PCR?

Answer 28

One for each strand of DNA

foreward and reverse

Question 29

How can a microarray be designed to show abnormal gene expression?

Answer 29

RNA solution + DNA probe
Cancer cells probe stained red
Normal cells probe stained Green.
Placed on same microarray, to show colour combinations.