Analysing Nucleic Acids

Question 1

give an example of how personalised medicine is helpful:

Answer 1

• over-expression of HER2 leads to more aggressive breast cancer - 20% of breast cancer is HER2 positive therefore more aggressive - drugs have been developed specifically for cancer where individuals have the HER2 gene - by sequencing the genome we can figure out if the person is HER positive or not

Question 2

Describe in viva cloning

Answer 2

1. Replicon and target DNA is cut with the same restriction endonuclease 2. Purify and Mix together 3. Join DNA fragments using DNA ligase 4. Transformation of recombinant DNA molecules into into host cell 5. Selective propagation on agar plate - use antibiotic resistance marker so only cells that take up replicon survive 6. expansion of cell culture Isolate target DNA

Question 3

what is a replicon?

Answer 3

it is a sequence capable of independent replication

Question 4

Describe type II restriction endonucleases

Answer 4

Enzymes that cut at a specific DNA sequence Recognition sequence is 4-8bp palindromic sequences they produce sticky or blunt end the longer the recognition site the less frequently it occurs in the DNA

Question 5

Describe electrophoresis

Answer 5

• DNA is negatively charged due to the phosphate backbone so moves to the anode when electrical force is applied - Smaller and more negatively charged fragments move further - after resolution the DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation

Question 6

What are hybridisation assays?

Answer 6

• Target DNA is immobilised and denatured - Probes (single stranded) bind to target DNA this is hybridisation as the complementary probes will bind

Question 7

What is southern blotting?

Answer 7

type of hybridisation - a method of transferring the electrophoresed fragments onto a membrane that immobilises them. - membrane can be washed with probes so that the probes can attach to complementary sequences. - Once the probes have annealed and excess probe has been washed off - membrane is exposed to a photographic film and kept in the dark - Bands will appear on the photographic film showing the positions of the fragments.

Question 8

Southern blot Northern blot Colony blot Tissue in situ Chromosome in situ Reverse hybridisation

Answer 8

Southern blot - DNA target and DNA probe Northern blot - RNA target and DNA probe Colony blot - bacterial DNA target and DNA probe Tissue in situ - RNA target and RBA probe Chromosome in situ - chromosome target and DNA probe

Question 9

Energy needed to denature DNA probe depends on…

Answer 9

Length - longer strand = more energy Base composition - more G-C = more energy Chemical environment - lots of 1+ charges outside can stabilise 1- charge of DNA

Question 10

What dies stringency mean?

Answer 10

How specifically the probe binds to target (low stringency means the probe can bind to the target with some degree of mismatch) (high stringency means in order for the probe to bind it must be a perfect match)

Question 11

Define melting temperature

Answer 11

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

Question 12

how does hybridisation of DNA fragments after separation work?

Answer 12

• after resolution the DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation - then mixed with labelled probes - can be detected by exposure to photographic film

Question 13

Hybridisation stringency is… and can be increased by …

Answer 13

The power to distinguish between related sequences Increase temperature and decrease Na+

Question 14

what temperature is hybridisation normally carried out at?

Answer 14

less than 25 degrees below the Tm Tm for mammal DNA is 87 degree

Question 15

what are examples of hybridisation?

Answer 15

Southern blot Northern blot Colony blot Tissue in situ Chromosome in situ Reverse hybridisation

Question 16

Describe in vivo cloning

Answer 16

Denature DNA (95 degrees) Anneal DNA with primer (50 degrees) Extend primer with DNA polymerase (72 degrees)

Question 17

What to consider when designing a primer

Answer 17

Length - 20 nucleotides for specificity Base composition - no tandem repeats = hairpins (hairpins are bad) Not complementary at 3’ end as primers join to each other instead (this would cause primer dimers)

Question 18

what is a DNA microarray?

Answer 18

A collection of microscopic DNA (or oligonucleotide) spots, commonly representing single genes, robotically arrays on a solid surface e.g. glass slide

Question 19

What are DNA microarrays used for?

Answer 19

Used to determine expression profile or many different genes at the same time

Question 20

what measurements are made from DNA microarrays?

Answer 20

Quantitative or Qualitative measurements can be made from microarrays which utilises the selective nature of hybridisation under high-stringency conditions

Question 21

how to denature a probe DNA?

Answer 21

• the denaturation of a probe DNA is achieved by heating until the hydrogen bonds between the bases break

Question 22

what is a polymerase chain reaction?

Answer 22

• this is an in vitro method that allows selective amplification of a specific target DNA within a heterogenous collection of DNA sequences

Question 23

what are applications of PCR?

Answer 23

• detecting point mutations - cDNA cloning - gene expression - DNA sequencing - DNA microarrays