DNA amplification

Question 1

describe pcr

Answer 1

DNA denatured
DNA cooled to enable hybridisation of primers
DNA heated to allow polymerisation

Question 2

pros and cons of Taq polymerase

Answer 2

can resist high temps - wont be denatured

however it doesnt proofread

Question 3

significance of AT’s low meling temp in PCR?

Answer 3

has low primer annealing temperature, hence non specific primer annealing can happen at low temps?

Question 4

significance of GC rich dsDNA?

Answer 4

leads to formation of a lot of secondary structures - needs secondary structure destabilisers

Question 5

describe probes

Answer 5

RNA complementary to mRNA product of a gene
ssDNA complementary to another ssDNA
can be labelled

Question 6

describe how PCR/cloning can be used in relation to breast cancer

Answer 6

BRCA1/2 genes produce tumour suppressing proteins; mutations lead to proteins not being produced, hence an increased likelihood of breast cancer.
primer made complementary to tumour sequence, PCR is done.

Question 7

describe how PCR/cloning can be used for paternal testing and foresnic science

Answer 7

short tandem repeats for paternal testing -1/2 should come from each parent
variable number of tandem repeats for criminal identification - should match to criminal
primer, PCR, electrophoresis.

Question 8

types of primers used in environmental science?

Answer 8

species specific primers - to determine presence of a particular species
polymorphic primers - to determine genetic diversity

Question 9

describe genomic library

Answer 9

whole genome cut by restriction enzymes
bacterial artifical chromosomes (plasmids) take up segements
collection of plasmids contianing the whole genome = genomic library

Question 10

describe cDNA library.

Answer 10

as per genomic library, except only exons are recorded.
mRNA reverse transcribed to ssDNA (poly T primer)
RNase nicks it
second strand formed

Question 11

describe microarrays

Answer 11

result in genome chips, with >5 million genetic probes placed on them.
computer analyses it

Question 12

two ways probes can be attached?

Answer 12

o Spotted arrays: attachment of probes prepared separately

o Oligonucleotide arrays: in situ synthesis of oligonucleotide probes

Question 13

how can microarrays be used for allelic screening?

Answer 13

probes could represent polymorphic variants in a population

Question 14

how can microarrays be used for comparative genome hybridisation

Answer 14

DNA from control and patient used

Question 15

how can microarrays be used to analyse gene expression?

Answer 15

mRNA reverse transcribed into ssCDNA, then attaches.