6- Analysis of nucleic acids

Question 1

What is hybridisation stringency

Answer 1

The the power to differentiate between related sequences

It increases with an
increased temperature and a decrease in sodium concentration

Question 2

What are type 2 restriction endonucleases

Answer 2

cleave DNA at specific recognition sequences
The recognition sites are usually at 4 8bp palindromic sequences
They can produce blunt or sticky ends

Question 3

Describe some hybridisation assays

Answer 3

Southern blot hybridisation(DNAtargetandDNAprobe)
Northern blot hybridisation(RNAtargetandDNAprobe)
o Colony blot hybridisation(bacterialDNAtarget,DNAprobe)
o Tissue in situ hybridisation (RNA target and RNA probe)
o Chromosome insitu hybridisation(ChromosometargetandDNAprobe)
o Reverse hybridisation – Microarrays (immobilised DNA or oligonucleotide probe, targetDNA solution)

Question 4

What does energy required to denature probe depend on

Answer 4

Strandlength(longer,morebondstobreak)
o Basecomposition(G Cpairhasthreebonds,A Thastwo)
o Chemical environment (monovalent cations stabilise the DNA duplex by neutralising charge, denaturants like formaldehyde or urea destabilise the DNA duplex)

Question 5

Describe how PCR is carried out

Answer 5

Heat to around 94 to denature
two primers, each complementary to the strand of DNA to be copied, are annealed to heat denatured DNA by lowering the temperature (50-60)
Thermostable TAQ polymerase and dNTPs extend the chain from the primers at 72
They are denatured again and the cycle is repeated many times using a PCR machine

There is s geometric increase in the amount of specific target sequence
The first one is seen after the third PCR cycle

Question 6

How should a primer for PCR be designed

Answer 6

20 nucleotides in length, so that there is specificity for the required target sequence
avoid tandem repeats to avoid hairpins and the melting temperature needs to be similar
The 3’ ends need to not be complementary to avoid primer dimers

Question 7

What are microarrays

Answer 7

A collection of microscopic oligonucleotide spots (representing single genes) on a side, used for expression profiling

High stringency conditions are used to make quantitive or qualitative measurements of expression of 1000s of genes simultaneously

Question 8

What is Tm

Answer 8

Measure of nucleic acid duplex stability

Question 9

How can nucleic acid analysis be used to tailor medicine

Answer 9

Over expression of HER2 protein leads to more aggressive breast cancer. Specific drugs can be given to target this mutation.

Question 10

How can DNA be cloned in vitro

Answer 10

Construct DNA molecules but cutting target DNA and replicant with restriction endonuclease
Transform DNA into host cells
Selective propagation of individual transformed cells
Expand cell culture