Nucleic acids 7- Analysis of nucleic acids

Question 1

Definition of Cloning:

Answer 1

A method of selectively amplifying DNA sequences of interest to generate homogenous DNA
populations

Question 2

Steps of Cell-based DNA cloning (in vivo)

Answer 2

1. cutting a target DNA and a replicon with restriction endonucleases, so that the ends of the two
   DNA sequences are compatible
2. mixing and joining the DNA fragments by using the enzyme DNA ligase
3. Transformation of the recombinant DNA molecules into host cells (bacteria, yeast)
4. Selective propagation of individual cell colonies on agar plate (selecting an antibiotic resistance marker in the
   replicon; only host cells with replicon and target DNA survive)
5. Expansion of the cell culture and isolation of recombinant DNA (using gene markers)

Question 3

Describe Restriction endonucleases (RE)

Answer 3

Type II Restriction Endonucleases are enzymes (usually dimers) that cleave DNA at specific recognition sequences.

Recognitions sequences are usually 4-8bp palindromic sequences.

They can produce blunt or sticky ends.

Used in cell based DNA cloning

Question 4

Describe the process of Electrophoresis

Answer 4

• DNA phosphate backbone has negative charge, therefore will move towards the anode (+VE) when an
  electrical force is applied
• When forced to move through a porous gel matrix (agarose / polyacrylamide gel) small fragments travel
  faster than larger ones
• After resolution, DNA can be isolated from the gel or transferred to a membrane to form a replica for
  hybridisation

Question 5

What is Nucleic acid hybridisation

Answer 5

• method for detecting specific nucleic acid sequences in which homologous single-stranded DNA or RNA
  molecules combine to form double-stranded molecules, i.e. takes advantage of the complementary nature
  of DNA/RNA
• involves a labelled nucleic acid probe to identify target homologous molecules in a mixture of nucleic acids

Question 6

What are the different types of hybridisation assays (6)

Answer 6

• Southern blot hybridisation (DNA target and DNA probe)
• Northern blot hybridisation (RNA target and DNA probe)
• Colony blot hybridisation (bacterial DNA target, DNA probe)
• Tissue in situ hybridisation (RNA target and RNA probe)
• Chromosome in situ hybridisation (Chromosome target and DNA probe)
• Reverse hybridisation – Microarrays (immobilised DNA or oligonucleotide probe, target DNA solution)

Question 7

What is denaturation

Answer 7

Denaturation of a probe DNA is achieved by heating until the hydrogen bonds between the bases holding
the two strands together are disrupted, leaving bases free to anneal with DNA probes

Question 8

What needs to be considered with the energy requirements for denaturation

Answer 8

• Strand length: longer strand = more hydrogen bonds to break
• Base composition: G-C pair has one more hydrogen bond than A-T, so harder to break
• Chemical environment: Monovalent cations (Na+) stabilise the DNA duplex by neutralising charge on
  phosphate backbone. Denaturants (formamide / urea) destabilise the DNA duplex, so less energy is required
  for separation

Question 9

What is melting temperature

Answer 9

Measure of nucleic acid duplex stability. It shows the Midpoint temperature of transition from double stranded (DS) to single stranded (SS) forms of nucleic acid (i.e. half-denaturation
point)

Question 10

What is Hybridisation Stringency and what does it increase with

Answer 10

The power to distinguish between related sequences increases with:

Increasing Temperature

Decreasing Na+ Concentration

Question 11

What is in vitro PCR

Answer 11

allows the selective amplification of a specific target DNA within a heterogenous collection of DNA sequences.

Question 12

Describe the process of in vitro PCR

Answer 12

Sequence information of the gene of interest in used to design primers complementary to the two strands
being copied (forward and reverse- approx 15-25 nucleotides in length)

 Primers are specifically annealed to heat-denatured DNA by lowering temperature
 Thermostable Thermophilus aquaticus (Taq) DNA polymerase extend 5’->3’ from the primers and generate
new strands using dNTPs (deoxyribonucleotide triphosphate)
 Taq polymerase comes from hot springs bacteria, so designed to work at high temperatures
 Leads to a massive increase in target sequence
 Denature = 94oC
anneal = 50-60 oC
extend = 72 oC
 Undergoes ~30 cycles, 20 hours. After 4 cycles half of the fragments are now target sequences

Question 13

What do you have to consider when designing a primer?

Answer 13

Length
- Usually about 20 nucleotides for a complex genomic DNA target – this gives the required specificity for
target sequence

 Base composition

• Avoid tandem repeats of nucleotides that can form hairpins
• %GC and length should give an ~equal Tm for each primer
• Otherwise one primer may bind without the other

 3’ end

• Avoid complementarity of the bases at the 3’ ends
• Primer dimers may result

Question 14

Applications of PCR

Answer 14

Detecting Point Mutations - restriction site changes, allele-specific amplification

cDNA cloning

Gene Expression - Reverse Transcription PCR

DNA Sequencing

DNA Microarrays