Nucleic acids 6- analysis of nucleic acids

Question 1

Describe the role of personalised medicine in HER-2 receptor positive breast cancer

Answer 1

Overexpression of HER2 leads to more aggressive breast cancer
(involved in cell growth and proliferation)
20% of breast cancer is Her2 positive- more aggressive
Drugs (biologic therapy) have been developed specifically for
cancer where individuals have a Her2 mutation
e.g. Trastuzumab (Herceptin)

Question 2

What are the clinical advantages of next generation sequencing

Answer 2

Allows us to introduce more targeted therapies- does the patient have the HER-2 mutation or not.
More practical- hand-held device

Question 3

What is DNA cloning

Answer 3

A method of selectively amplifying DNA sequences of interest to generate homogenous DNA populations

Cell-based DNA cloning (in vivo)

Cell-free DNA cloning (in vitro) - PCR

Question 4

What is a replicon

Answer 4

A sequence capable of independent replication (a plasmid, bacteriophage, Yeast Artificial Chromosome)

Question 5

Why do we need to cut the genome into fragments before cloning

Answer 5

The genome is too long and unwieldy to be handled easily in the laboratory- hence it needs to be cut into more manageable pieces.

Question 6

Describe cell-based DNA cloning

Answer 6

cutting a target DNA and a replicon, with specific enzymes, restriction endonucleases, so that the ends of the two DNA sequences are compatible.mixing and joining the DNA fragments by using the enzyme DNA ligase. Transformation of the recombinant DNA molecules into host cells (bacteria, yeast.Selective propagation of individual cell colonies on agar plate.selectable antibiotic resistance marker in the replicon; only cells with replicon survive)
Expansion of the cell culture and isolation of recombinant DNA- culture in a growth medium.

Question 7

What is the role of the replicon in DNA cloning

Answer 7

It is the vector-

Question 8

What are restriction endonucleases

Answer 8

Type II restriction endonucleases are enzymes that cleave DNA at specific recognition sequences
Recognition sites are usually 4-8bp palindromic sequences

Question 9

Describe the two types of cut that restriction endonucleases make

Answer 9

Blunt- cut at the point of symmetry
Staggered cuts- create sticky ends- short, single-stranded overhangs that help the cut DNA molecules join back together by Complementary base paring with, with the aid of ATP and DNA ligase to reseal the sugar phosphate-backbone.

Question 10

Where are restriction endonucleases obtained from

Answer 10

Restriction endonucleases are one half of bacterial restriction-modification systems, a kind of primitive immune system. Host DNA is protected by methylation of a base in the RE site by a specific methylase - RE will only cleave unmethylated DNA from invading organisms, not host DNA

Question 11

If the recognition site is longer is it likely to occur more frequently or less frequently within the genome

Answer 11

Less frequently

Question 12

How are DNA fragments separated and how does this work

Answer 12

Electrophoresis

DNA is negatively charged due to its phosphate backbone and it moves towards the anode (+ve electrode) when an electrical force is applied to a DNA solution

Question 13

Describe how the DNA fragments are separated based on size

Answer 13

DNA size resolution

When DNA is forced to travel through a porous gel matrix (agarose / polyacrylamide gel) small fragments are retarded less than large fragments and hence travel faster

Question 14

How do we isolate the desired fragment

Answer 14

Excise it with a scalpel or blade

Question 15

How can we visualise DNA fragments

Answer 15

Expose the gel to a dye that fluoresces under UV light when it is bound to DNA- when placed in a UV box, the individual bands glow bright orange, or bright white when the gel is photographed in black and white.
Incorporate a radioisotope- beta particles emitted from the P-32 can activate the radiation-sensitive particles in photographic film, a sheet of film placed on top of the agarose gel will, when developed, show the position of all the DNA bands..

Question 16

What is the purpose of nucleic acid hybridisation

Answer 16

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

Question 17

What is a standard assay in hybridisation

Answer 17

Standard assay involves a labeled ( * ) nucleic acid probe to identify homologous related target molecules in a mixture of unlabeled nucleic acids

Question 18

Describe southern blot hybridisation

Answer 18

Target DNA (e.g. from an agar plate or gel) is immobilised on a solid support - nylon or nitrocellulose membrane - which readily binds single-stranded nucleic acid (e.g. denatured DNA or mRNA) and then hybridised with a solution of (radioactively or fluorescently) labeled (*) probe

Question 19

How is the DNA denatured in southern blotting

Answer 19

By the presence of an alkali solution, buffer, drawn towards paper towels, carrying the singl-stranded target DNA to the nitrocellulose paper where it is then immobilised.

Question 20

Why do we wash the nitrocellulose sheet after adding the probe

Answer 20

So that only probe molecules that have hybridised to the DNA on the paper remain attached.

Question 21

Describe the variation in hybridisation assays

Answer 21

Southern blot hybridisation (DNA target and DNA probe)
(after Ed Southern)
Northern blot hybridisation (RNA target and DNA probe)
Colony blot hybridisation (bacterial DNA target, DNA probe)
Tissue in situ hybridisation (RNA target and RNA probe)
Chromosome in situ hybridisation (Chromosome target and DNA probe)

Reverse hybridisation – Microarrays (immobilised DNA or oligonucleotide probe, target DNA solution)

Question 22

Describe the purpose of in situ hybridisation

Answer 22

Single-stranded DNA or RNA labelled probes detect complementary DNA or RNA within a tissue or cell, or even on a chromosome, showing which genes are expressed.

Question 23

How do we denature probe DNA, and what does the energy to do this depend on

Answer 23

Denaturation of a probe DNA is achieved by heating until the hydrogen bonds between the bases holding the two strands together are disrupted.

The energy needed to do this depends on:

Strand length: longer strand = more hydrogen bonds to break

Base composition: G-C pair has one more hydrogen bond than A-T, so harder to break

Chemical environment:
Monovalent cations (Na+) stabilise the DNA duplex by neutralising charge on phosphate backbone
denaturants (formamide / urea) destabilise the DNA duplex

Question 24

What is the melting temperature (Tm)

Answer 24

Melting temperature (Tm) - measure of nucleic acid duplex stability.
Midpoint temp. of transition from double stranded (DS) to single stranded (SS) forms of nucleic acid

For mammalian genomic DNA (40% GC) this is ~87oC

Hybridisation is carried out at temperatures <25oC below Tm- to ensure a 50:50 mix of DS and SS DNA, so that hybridisation can occur.

Question 25

What is hybridisation stringency

Answer 25

Hybridisation stringency (i.e. the power to distinguish between related sequences) increases with:
Increase in temperature
Decrease in Na+ concentration.
reaction conditions, notably temperature and salt, that dictate the annealing of single-stranded DNA/DNA, DNA/RNA, and RNA/RNA hybrids
at high stringency, duplexes form only between strands with perfect one-to-one complementarity
lower stringency allows annealing between strands with some degree of mismatch between bases

Question 26

Describe cell-free DNA cloning

Answer 26

Polymerase Chain Reaction (PCR)

In vitro method to allow selective amplification of a specific target DNA within a heterogeneous collection of DNA sequences (e.g. total genomic DNA or complex cDNA population)

Question 27

What are the three requirements for PCR.

Answer 27

Some sequence information of the target (e.g. gene of interest) is needed to design 2 primers (15 - 25 nucleotides in length), one complimentary to each strand of the DNA to be copied (“forward” and “reverse”)
DNA polymerase can only extend and existing chain. We need two primers, as chain extension can only proceed in 5’-3’.
We need the two strands of the DNA double helix to be anti-parallel

Question 28

What are the advantages of PCR

Answer 28

Quicker, can amplify even tiny samples- useful in crime scene
Selective- although original irrelevant DNA is still present, the cloned DNA is in such an excess that we assume the final product to be a slightly impure preparation of the target sequence

Question 29

Describe the process of PCR

Answer 29

Primers are specifically annealed to heat denatured (95) DNA by lowering temperature (50-60)

Thermostable Thermophilus aquaticus DNA polymerase + dNTPs extend 5’->3’ from the primers and generate new strands- 72- optimum temp for Taq Polymerase.
This cycle is then repeated around 30 times

Question 30

What does the selectivity of PCR depend on

Answer 30

Having primers that will only anneal to the desired target. This means that having short primers is ideal, and fine-tuning the temperature is essential, too low and the primers may be able to hybridise with less stringency- computer programmes assist with primer design to ensure that they are only complementary to the target region of DNA.

Length
Usually about 20 nucleotides for a complex genomic DNA target – gives required specificity for target sequence

Base composition
Avoid tandem repeats of nucleotides that can form hairpins
%GC and length should give an ~equal Tm for each primer

3’ end
Avoid complementarity of the bases at the 3’ ends
Primer dimers may result

Question 31

What are the applications of PCR

Answer 31

Typing genetic markers (Restriction Fragment Length Polymorphisms)

Detecting point mutations
restriction site changes, Allele-specific amplification

cDNA cloning

Genome walking

Gene expression – Reverse Transcription-PCR (reverse transcribe mRNA to DNA using oligo-dT [binds to polyA tail], then amplify)

Introducing mutations experimentally (base mismatches)

DNA sequencing

DNA microarrays

Question 32

What are DNA (oligonucleotide) arrays

Answer 32

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

Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridisation under high-stringency conditions, with fluorophore-based detection

DNA arrays are commonly used for expression profiling, i.e. monitoring expression levels of thousands of genes simultaneously, or for comparative genomic hybridization

Question 33

Why doesn’t the target sequence rehybridize to the original sequence

Answer 33

it may, but the primers are present in much greater numbers.

Question 34

What are the applications of microarrays

Answer 34

mRNA or gene expression profiling
- monitoring expression levels for thousands of genes simultaneously is relevant to many areas of biology and medicine, such as studying treatments, disease, and developmental stages. For example, microarrays can be used to identify disease genes by comparing gene expression in diseased and normal cells

SNP detection arrays - looking for Single Nucleotide Polymorphisms in the genome of populations

Question 35

Describe simply the process of microarray

Answer 35

cDNA probe binds to mRNA from cell- disadvantage of only showing expression of genes targeted by the probe- different colours represent different levels of gene expression- reverse hybridisation.