Molecular biology techniques

Question 1

what methods can we use to determine which genes are responsible for inherited disorders

Answer 1

(order of low resolution to high)

linkage analysis- family studies

DNA hybridisation studies- southern blotting

modern molecular techniques-PCR

nt sequencing

microarrays

NGS, bioinformatics to analyse whole genome sequence

Question 2

in gel electropheresis, restricted fragments are seperated by…

Answer 2

SIZE

- compared with standard markers

Question 3

difference in usage of polyacrylamide gel and when you would use agarose gel

Answer 3

polyacrylamide gels used to separate LOW molecular weight molecules

agarose gels used to separate HIGH molecular weight molecules

Question 4

what are plasmids

Answer 4

1. extra-chromosomal DNA molecules
2. confer antibiotic resistance
3. vectors= plasmids used specifically for gene cloning

Question 5

3 basic requirements of vectors

Answer 5

1. ability to self-replicate within host
2. possession of selection marker
3. must be capable of being digested

Question 6

what is a primer

Answer 6

short oligo-nucelotide sequence- 20-35 nt that will anneal to the 3’ portion of DNA template

Question 7

temp of key stages in PCR

Answer 7

denaturation: 94-96°C
annealing: 68°C
elongation: 72°C

Question 8

advantages of southern blotting- DNA hybridisation

Answer 8

1. detect copy no. of gene
2. detect gross alterations in gene
3. can visualise specific DNA fragment among thousands
4. can probe for MORE THAN ONE FRAGMENT of DNA

Question 9

outline key steps of DNA hybridisation/ southern blot

Answer 9

Take the digested DNA with appropriate restriction enzyme. Run fragments on electrophoresis gel to separate. Denature DNA to make single strands and transfer to nitrocellulose membrane. Hybridise DNA with specific probe. Detect DNA fragment

Question 10

how may the probe be labelled in DNA hybridisation/ southern blot

Answer 10

1. 32P label
2. enzymatic label

both allow colorimetric detection

Question 11

difference between northern blot and southern blot

Answer 11

northern blot= rna hybridisation

Southern blot= dna hybridisation

Question 12

key basis for Sanger sequencing

Answer 12

1. the use of DI-DEOXYnt. which does NOT have OH group on the THIRD CARBON of SUGAR MOIETY OF NT.

thus when incorporated into growing DNA chain, cannot be extended= chain termination

2. 4 reactions happen in parallel, all labelled with a different colour FLUOROCHROME
3. detected on a CHROMOATOGRAM which shows the first chain to be terminated as first peak

Question 13

Sanger sequencing has since been replaced by NGS. outline the advantage of NGS over Sanger

Answer 13

1. modern array based parallel automatised sequencing method supported by bioinformatics, data processing
2. faster speed- genomes sequenced within days
3. better fidelity (accurate dna polymerase)
4. better cost

Question 14

what are the benefits of genetic screening

Answer 14

Genetic differences in drug metabolism identified to maximise therapeutic benefits (personalised medicine).

Adverse drug reactions – at the moments these seem idiosyncratic but there is a scientific basis.

Different responses to medicines – can we use genetic predictors?

Identify “at risk groups” and suggest lifestyle changes.

Question 15

risks of genetic screening

Answer 15

MASS screening is expensive

misuse of data

socially stigmatising

Will individuals react inappropriately to test results – depression/suicide for negative findings or even ignoring health warnings, smoking and drinking more if “good genes” identified.

may lead to selection of favoured genes within population= loss of diversity. diversity is a PROTECTIVE MECHANISM for a species (our ability to adapt/ survive pandemic contagious disease) E.g. sickle cell aneamia gene protects against malaria- balanced polymorphism