Molecular Analysis

Question 0

What is electrophoresis? What is the purpose of the different components? What is the difference between using DNA or proteins?

Answer 0

Separates macromolecules based on size and charge.

Molecules migrate due to electric charge placed on gel. Smaller the fragment, the further it migrates whilst the current is applied.

Gel: matrix allows fragments to pass through (DNA = agarose gel, proteins = polyacrylamide gel)

Buffer: allows charge on DNA samples, proteins must be coated with the detergent SDS to give them an even negative charge (size only factor in migration)

Power supply: generates charge difference across gel

Stain: identifies presence of separated DNA (ethidium bromide) or proteins

SDS-PAGE: beta-mercaptoethanol (reducing agent) denatures proteins to leave only primary structure

Question 1

What are the features of restriction enzymes? How do we differentiate our DNA from foreign DNA?

Answer 1

Recognise and cut dsDNA at specific points (restriction sites)

Endonucleases

Restriction sites mainly palindromes

Methylation protects our DNA from being cleaved for degradation

Question 2

What is restriction analysis?

Answer 2

Usage of restriction enzymes to compare samples of DNA e.g. to identify mutations in the DNA sequence which leads to deletion/addition of restriction sites.

Investigates: size of DNA fragments, mutations, DNA variations,

Can be used to clone DNA

Question 3

Outline the process of gene cloning.

Answer 3

1) Use restriction endonucleases to cleave gene of interest and DNA ligase to seal the gene into the vector (e.g. plasmids: can replicate independently as the origin of replication is present)
2) Insert vector into host (e.g. transduction via virus, translocation, conjugation etc.)
3) Identify and isolate hosts which have successfully picked up the recombinant vector DNA (transformed bacteria) by screening e.g. antibiotic resistance by inserting cloned gene into plasmid antibiotic gene
note: can use reverse transcriptase to convert mRNA into cDNA

Question 4

Outline the PCR reaction and its purpose.

Answer 4

Amplifies small fragments of DNA using a DNA or RNA template for further experimental procedures.

Forward and reverse primers define region to be copied (target DNA)

1) DNA denatured
2) Primers anneal
3) Taq polymerase (Thermus aquaticus) extends primers

25-30 cycles, exponential increase in target DNA

Question 5

What is isoelectric focusing?

Answer 5

Proteins separate on basis of charge (migrate through pH gradient until pH=pI)

No net charge at pI, therefore migration stops

Question 6

What is 2D-PAGE?

Answer 6

SDS-PAGE + IEF

Allows separation of complex mixtures of proteins (e.g. if proteins have the same Mr/pI)

Question 7

How do you identify proteins?

Answer 7

Proteomics = analysis of all proteins expressed from a genome

1) Digest protein with trypsin (recognises specific aa)
2) Mass spectrometry
3) Generate list of peptide sizes
4) Compare to database of predicted peptide sizes to identify proteins

Question 8

What is the difference between monoclonal and polyclonal antibodies?

Answer 8

MONOCLONAL:

• produced by 1 B-lymphocyte
• 1 identical antibody (therefore specific to 1 antigen, and recognises 1 epitope only)

POLYCLONAL:

• produced by many B-lymphocytes
• multiple different antibodies
• specific to 1 antigen (but attack it in different ways)
• multiple epitopes

Question 9

Outline the process of DNA sequencing.

Answer 9

Dideoxynucleoside triphosphates (ddNTPs) prevent elongation when incorportated (as there is no 3’-OH)

Add fluorescent ddNTPs to denatured template with DNA pol

Produces a range of fragments ending in fluorescent bases which are read by a computer, which interprets the overlapping fragments

Question 10

Outline Western blotting.

Answer 10

Separation of proteins by electrophoresis.

Transfer to nitrocellulose/nylon filter

Detect proteins bound to antibodies which fluoresce (produces immunoblot)

Question 11

What is the difference between Southern, Northern, and Western blotting?

Answer 11

SOUTHERN = DNA

NORTHERN = RNA

WESTERN = proteins

Question 12

Outline the function/mechanism of ELISA.

Answer 12

Enzyme-linked immunoabsorbent assay (work out concentration of proteins)

Antigen-coated well + specific antibody + enzyme-linked antibody

= substrate added which is converted to a coloured compound which is proportional to the amount of specific antibody

Radioimmunoassay = radiolabelled primary antibody

Enzyme assay = work out enzyme kinetics e.g. by spectrophotometry

Question 14

Outline Southern blotting.

Answer 14

1) Digest DNA using restriction enzymes
2) Separate DNA fragments by gel electrophoresis
3) Transfer DNA fragments to nylon or nitrocellulose (DNA bound by heat/UV)
4) Hybridise filter with labelled gene probe
5) Detect hybridisation by exposure of filter to X-rays

Question 15

What is DNA fingerprinting?

Answer 15

Repeated, non-coding sequences of DNA (microsatellites).

Number of repeats varies between individuals

Can determine family relationships

Question 16

What is DNA profiling?

Answer 16

Variation in no. of copies of small tandem repeats between individuals.

Used for forensics.

Question 17

What is a microarray? What are the advantages of using a microarray?

Answer 17

Genome-wide analysis of mRNA shows conditional expression of mRNA.

Always compares two genomes.

Black = no hybridisation 
Purple = both genomes expressed in equal amounts 
Red = healthy mRNA expressed only 
Green = tumour mRNA expressed only

Can investigate thousands of genes simultaneously

Question 18

What is array comparative genome hybridisation?

Answer 18

Extract and label DNA from two genomes (Green = normal individual, Red = patient)

Mix and microarray

Work out the red:green

Deletion in patient mRNA = less red

Duplication in patient mRNA = more red

Question 19

What is reverse-transcriptase PCR? What differentiates PCR and RT-PCR?

Answer 19

Use polyA tail to isolate mature mRNA from rRNA and tRNA (mRNA used as it has no introns).

Use reverse transcriptase to make a mRNA-cDNA complex

Digest mRNA using RNase and then use primers to synthesise new cDNA strand

As no introns are present, RT-PCR will amplify different sequences to PCR

Question 20

What is nested PCR?

Answer 20

Second round of amplification of PCR product

Primers used are specific to a section of the fragment, therefore a shorter sequence is amplified (more specific)

Question 21

What is the effect of using allele-specific primers in PCR?

Answer 21

Primers specific to an allele

Overhang at 3’ end will prevent elongation and will not produce a PCR product

Question 22

List and describe some of the chromosome analyses. What might these analyses reveal?

Answer 22

Karyotyping = chromosomes isolated, stained, and ordered

Fluorescence in situ hybridisation (FISH) = fluorescent DNA probe hybridises with denatured DNA which is then condensed into the chromosome

Chromosome painting = use many different fluorescent probes for many different locations on a chromosome in order to colour-code the chromosomes

Anaphase lag affecting the chromosome number.
Translocations
Large deletions/duplications

Question 25

What is the definition of hybridisation? What is the definition of a heteroduplex?

Answer 25

DNA hybridisation = process of forming a dsDNA molecule from two complementary strands of DNA annealing

Heteroduplex = dsDNA formed by the recombination of complementary strands of DNA from different sources

Question 26

When is PCR not helpful? What could be used instead?

Answer 26

Expansion repeats unreliably amplified, causing collapse of fragments, leading to underestimation of allele lengths

e.g. with Huntington’s, Fragile X syndrome

Use Southern blotting

Question 27

When would you use Southern blotting?

Answer 27

• large deletions/duplications
• variations
• gene expansion/triplet expansions

Question 28

When would you use ELISA?

Answer 28

Measure concentrations of serum proteins e.g. cortisol, insulin