Analysis of Nucleic acids

Question 1

DNA cloning

Answer 1

Selective amplification of DNA sequences of interest to generate homogenous DNA populations
Can be done in vivo or in vitro

Question 2

Describe in vivo cloning

Answer 2

1. Replicon and target DNA is cut with the same restriction endonuclease (so ends are compatible)
2. Mix together
3. Join using DNA ligase
4. Transform recombinant DNA into host cell e.g. yeast
5. Selective propagation of individual colonies on agar plate- use antibiotic resistance marker so only cells that take up replicon survive
6. Expansion of cell culture and isolation of recombinant DNA

Question 3

Describe type II restriction endonucleases

Answer 3

Enzymes that cleave DNA at specific recognition sequences
Recognition sites are 4-8bp palindromic sequences
Sticky or blunt ends

Question 4

What is the result of host DNA being protected by methylation of a base in the RE site by a specific methylase?

Answer 4

Restriction endonuclease will only cleave un-methylated DNA from invading organisms, not host DNA

Question 5

Describe electrophoresis

Answer 5

DNA is negatively charged due to phosphate backbone
so moves to the anode (+ve electrode)
Smaller and more negatively charged fragments move further

Question 6

What is electrophoresis used for?

Answer 6

Separation of DNA fragments

Question 7

What happens after resolution in electrophoresis?

Answer 7

DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation

Question 8

What is nucleic acid hybridisation?

Answer 8

method for detecting specific nucleic acid sequences

Homologous single-stranded DNA or RNA molecules combine to form double-stranded molecules

Question 9

Labelling in nucleic acid hybridisation

Answer 9

Standard assay involves a labeled nucleic acid probe to identify homologous related target molecules in a mixture of unlabelled nucleic acids

Question 10

What occurs in hybridisation assays?

Answer 10

Target DNA is immobilised on a solid support (e.g. nylon) which readily binds ss nucleic acid (denatured DNA/ mRNA)
Then hybridised with solution of labelled probe

Question 11

Hybridisation blotting of DNA fragments after separation

Answer 11

After resolution, DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation with a labelled probe detected by exposure of photographic film

Question 12

What is southern blotting?

Answer 12

Making electrophoresis fragments into immobilised hybridisation ones.
Can be washed with probes and then probes washed away
Can use photographic film to see where the bands are

Question 13

Types of hybridisation assay

Answer 13

Southern blot: DNA target + DNA probe
Northern blot: RNA target + DNA probe
Colony blot: bacterial DNA target + DNA probe
Tissue in situ: RNA target + RNA probe
Chromosome in situ: chromosome target + DNA probe

Question 14

What does reverse hybridisation involve?

Answer 14

Microarrays (immobilised DNA or oligonucleotide probe, target DNA solution)

Question 15

What can Gene specific probes be used for?

Answer 15

To detect restriction fragment length polymorphisms and identify organisms genotype

Question 16

What can in situ hybridisation be used for?

Answer 16

To locate specific genes on chromosomes

Question 17

How do you denature a probe DNA?

Answer 17

Heat until hydrogen bonds between bases holding 2 strands are disrupted

Question 18

Energy needed to denature DNA probe depends on…

Answer 18

Length: longer strand = more hydrogen bonds
Base composition: GC pair has 1 more hydrogen bond than AT, so harder to break
Chemical environment: monovalent cations stabilise DNA duplex by neutralising charge on phosphate backbone
denaturants destabilise DNA duplex

Question 19

Define melting temperature

Answer 19

The midpoint temperature of transition between double stranded to single stranded nucleic acids

Question 20

Melting temperature for mammalian genomic DNA and temperature hybridisation is carrier out at

Answer 20

Tm= 87 degrees C

Hybridisation is carried out at <25 degrees below Tm

Question 21

What is hybridisation stringency?

Answer 21

The power to distinguish between related sequences

Question 22

What does hybridisation stringency increase with?

Answer 22

Increase in temperature

Decrease in Na+ concentration

Question 23

What occurs at high stringency?

Answer 23

Duplexes form only between strands with perfect 1-to-1 complementarity

Question 24

What occurs at low stringency?

Answer 24

Annealing between strands with some degree of mismatch between bases

Question 25

What is the in vitro method of DNA cloning?

Answer 25

Polymerase chain reaction (PCR)

Question 26

Describe PCR methodology

Answer 26

Denature DNA (94 degrees)
Anneal DNA with primer (50-60 degrees)
Extend primer with DNA polymerase (72 degrees)
= Thermostable Taq polymerase added with dNTPs to extend 5’ to 3’ from primers to generate new stands

Question 27

What to consider when designing a primer

Answer 27

Length: 20 nucleotides, gives required specificity
Base composition: no tandem repeats that can form hairpins
Avoid complementary at 3’ end: as primers join to each other instead (primer dimers)

Question 28

What needs to be designed before PCR can be carrier out?

Answer 28

2 primers

1 complimentary to each strand of the DNA to be copied

Question 29

Name 5 applications of PCR

Answer 29

Typing genetic markers
Detecting point mutations
DNA sequencing
DNA microarrays
Genome walking

Question 30

What is a DNA (or oglionucleotide) microarray?

Answer 30

a collection of microscopic DNA (or oligonucleotide) spots, commonly representing single genes, robotically arrayed on a solid surface

Question 31

What do qualitative or quantitative measurements with DNA microarrays utilise?

Answer 31

the selective nature of DNA-DNA or DNA-RNA hybridisation under high-stringency conditions, with fluorophore-based detection

Question 32

What are DNA microarrays used for?

Answer 32

Used for expression profiling

To monitor expression levels of 1000s of genes simultaneously or comparative genomic hybridisation

Question 33

How can microarrays be used to identify disease genes?

Answer 33

By comparing gene expression in diseased and normal cells