Analysis of Nucleic Acids

Question 1

Summarise in vitro DNA cloning

Answer 1

target DNA and replicon cut out using same restriction endonuclease, mix and join with ligase, transform recombinant DNA into host cells, selective propagation of cell colonies on agar plate using marker (e.g. antibiotic resistance), expand and isolate cell culture

Question 2

How do T2 restriction endonucleases work?

Answer 2

cleave DNA at specific 4-8 bp palindromic recognition sequence

Question 3

How are host cells protected from the restriction endonuclease?

Answer 3

methylation of base at recognition sequence and only unmethylated cleaved

Question 4

Name 2 fragment seperation techniques

Answer 4

electrophoresis and DNA size resolution

Question 5

Describe DNA size resolution

Answer 5

DNA forced to travel through porous gel matrix, small fragment travel faster, DNA isolated from gel or transferred to membrane to form replica for hybridisation with labelled probe

Question 6

What is nucleic acid hybridisation?

Answer 6

method for detecting specific nucleic acid sequences in which homologous single stranded DNA/RNA molecules combine to form double stranded

Question 7

Describe the hybridisation assay process

Answer 7

target DNA immobilised on solid support which readily binds to single stranded nucleic acid (denatured DNA/mRNA) and then hybridised with a solution of labelled probe

Question 8

What is the effect of the chemical environment on denaturation?

Answer 8

monovalent cations stabilise DNA duplex due to phosphate, denaturants (urea/formamide) destabilise duplex

Question 9

What is a measure of nucleic acid duplex stability?

Answer 9

melting temperature

Question 10

What is hybridisation stringency?

Answer 10

ability to distinguish between related sequences

Question 11

What does high stringency mean?

Answer 11

duplexes only form between strands with perfect complementarity

Question 12

Define PCR

Answer 12

in vitro method to allow selective amplification fo a specific sequence of target DNA within a collection of sequences

Question 13

Summarise PCR methodology

Answer 13

denture DNA, 2 primers required (one for each DNA strand), primers anneal to denatured DNA by lowering temperature, thermostable Taq DNA polymerase and dNTPs extend from the primers and generate new strands

Question 14

What temperature is required for denaturing, annealing and extending?

Answer 14

94, 50-60, 72

Question 15

How long should a PCR primer be? Why?

Answer 15

20 nucleotides as gives required specificity

Question 16

What should be avoided in PCR primer? Why?

Answer 16

tandem repeats as can form hairpins

Question 17

Why should complementarity of 3’ end bases be avoided?

Answer 17

forms primer dimers

Question 18

Name some PCR applications

Answer 18

detecting point mutation, cDNA cloning, genome walking, DNA sequencing

Question 19

What are DNA microarrays?

Answer 19

collection of microscopic DNA spots, representing single genes arranged on a solid surface

Question 20

What are DNA microarrays used for?

Answer 20

expression profiling, SNP detection arrays

Question 21

Expression profiling use

Answer 21

monitor gene expression levels, relevant for stiff of disease treatments and development

Question 22

What are SNP detection arrays?

Answer 22

look for single nucleotide polymorphisms in population genome