NA hybridisation in molecular diagnostics

Question 1

define NA hybridisation

Answer 1

binding 2 pieces of ssNA to one another via Hbond in b/w bases

Question 2

Which pair of nucleotides require more energy to break?

Answer 2

• A-T: 2x H bond
• G-C: 3x Hbond
* G-T = Hi temp to break

Question 3

How does southern blotting work? (process0

Answer 3

1. Genomic DNA digested by R.digest
2. Fragments separated on agarose gel
3. photo of gel is taken = band s visualised
4. DNA may be depurinated w/ 0.2M HCl
5. DNA denatured by 0.4naOH
6. NaOH neutralised w/ Tris-HCl & NaCl
7. Transfer begins
8. membrane incl=ubated in pre-hybridiation solution
9. membrane incubated w/ probe - hybridizes w target of intrest

Question 4

How does NA lenght affect NA hybridisation?

Answer 4

• ≥20bp in length for specificity
• Longer NA more stable once hybridized
• Avoid regions that are compliment bc form 2º structures

Question 5

How does mismatch position affect NA hybridisation?

Answer 5

mismatch @ center of probe = more disruption

Question 6

How does hybridisation temp affect NA hybridisation?

Answer 6

• the melting temp (Tm) of NA = the temp @ which 50% of NA is denatured
• Dec. rxn temp = DNA hybridisation but dec specificity
• INc rxn temp = DNA denaturation but inc. specificity

Question 7

How does salt [ ] affect NA hybridisation?

Answer 7

• neg charge phosphate groups repel each other & os overcome by H bonding
• monovalent cations (Na+) decrease repulsive force = hybridisation = dec specificity
• dec. salt [ ] = decreases hybridisation = inc. specificity = allow homologous binding

Question 8

Difference b/w northern blot & southern blot

Answer 8

• Northern blot detect RNA

- Southern blot detect DNA

Question 9

Describe microarray analysis

Answer 9

1. probes spotted onto glass
2. Fragmented & fluorescnt Test DNA added to slide = hybridize
3. Unbound test DNA is washed off
4. Hybridised DNA detected via fluorescence

Question 10

microarray analysis is used for….

Answer 10

• Mutation detection (SNP analysis)

* Gene expression studies

Question 11

If we were using microarray analysis to determine if 2 samples have similar gene expression isolate _ from the sampls

Answer 11

mRNA

Question 12

Interpretation of microarray analysis

Answer 12

• If red dye = reference
• If green dye = test
  • if more red= expression higher reference = decreased expression in test
  • If yellow = equal in both samples
  • If more green = expression higher test = inc expression in test

Question 13

Describe bead arrays

Answer 13

• different probe attached to specifically coloured bead
• DNA fluorescently labelled a diff colour from the beads
  = so if DNA hybridizes on bead both colours (from DNA & bead) will light up

Question 14

microarray analysis is used to….

Answer 14

detect SNP