6 - Analysis of nucleic acids

Question 1

What is the cancer genome and stratified medicine?

Answer 1

An example of using DNA technology to provide treatment targeted to individual patients. (Herceptin for treatment of HER2 positive breast cancer)

• Stratified medicine
• Genome-wide analysis studies
• Next generation sequencing; the $1000 genome

Question 2

How are recombinant DNA molecules constructed in vitro? What 2 enzymes are required?

Answer 2

Restriction endonuclease cut a target DNA and a replicon, so that the ends of the two DNA sequences are compatible. DNA ligase joins the DNA fragments.

Question 3

What is done with recombinant DNA?

Answer 3

Transformation of the recombinant DNA molecules into host cells (bacteria, yeast).
Selective propagation of individual cell colonies (selectable antibiotic resistance markers).
Expansion of the cell culture and isolation of recombinant DNA.

Question 4

What are restriction endonucleases?

Answer 4

Enzymes that cleave DNA at specific recognition sites, usually 4-8bp palindromic sequences – produce “blunt” or “sticky” DNA ends.
The longer the recognition site, the less frequently it occurs in DNA.

Question 5

How are DNA fragments separated?

Answer 5

Electrophoresis – DNA is negatively charged due to its phosphate backbone and moves towards the anode (+ve electrode) when an electrical force is applied to it.
When DNA is forced to travel through a porous gel matrix (agarose / polyacrylamide gel) small fragments are retarded less than large fragments and hence travel faster.
After resolution, DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation.

Question 6

What is Nucleic Acid Hybridisation?

Answer 6

Method for detecting specific nucleic acid sequences in which homologous single-stranded DNA or RNA molecules combine via homologous base-pairing to form double-stranded molecules.
Standard assay involves a labeled nucleic acid probe (DNA, RNA or oligonucleotide) to identify homologous related molecules in a mixture of target unlabeled nucleic acids.

Question 7

What are Hybridisation assays?

Answer 7

Target DNA is immobilised on a solid support – nylon or nitrocellulose membrane – which readily binds single-stranded nucleic acid (e.g. DNA) and then hybridised with a solution of labeled probe (radioactive or fluorescent).

Question 8

Examples of hybridisation assays?

Answer 8

Southern blot hybridisation (DNA target and DNA probe) Finds identity of DNA, size and abundance.
Northern blot hybridisation (RNA target and DNA probe)
Colony blot hybridisation (bacterial DNA target, DNA probe)
Chromosome in situ hybridisation (Chromosome target and DNA probe)
Tissue in situ hybridisation (RNA target and RNA probe)
Reverse hybridisation – Microarrays (immobilised DNA or oligonucleotide probe, target DNA solution)

Question 9

What is Melting temperature and hybridisation stringency?

Answer 9

Denaturation of a probe DNA = heating until the hydrogen bonds holding the two strands together are disrupted.
The energy needed to do this depends on strand length (longer strand = more hydrogen bonds to break), base composition (G-C pair has one more hydrogen bond than A-T) and chemical environment (monovalent cations stabilise the DNA duplex by neutralising charge on phosphate backbone; denaturants (formamide/urea) destabilise the DNA duplex).
Melting temperature (Tm) – measure of nucleic acid duplex stability (Hybridisation is carried out at temperatures < 25oC below Tm).
Hybridisation stringency (i.e. the power to distinguish between related sequences) increases with increase in temperature and decrease in salt concentration.

Question 10

What is Cell-free DNA cloning – Polymerase Chain Reaction (PCR)?

Answer 10

In vitro method to allow selective amplification of a specific target DNA within a heterogeneous collection of DNA sequences (e.g. total genomic DNA or complex cDNA population).

Question 11

How does PCR work?

Answer 11

1. DNA heated and is denatured.
2. Some sequence information is needed to design 2 primers (15 – 25 nucleotides in length), one complimentary to each strand of the DNA to be copied. Primers are specifically annealed.
3. Thermostable DNA polymerase + dNTPs extend from the primers and generate new strands.
4. Denature the DNA and repeat the cycle many times -> geometric increase

Question 12

Applications of PCR?

Answer 12

Typing genetic markers; Detecting or introducing point mutations; cDNA cloning; genome walking; detecting gene expression; DNA sequencing, qPCR