Molecular Techniques - DNA

Question 1

Which technique is used to amplify DNA?

Answer 1

Polymerase Chain Reaction

Question 2

What components are required for PCR?

Answer 2

1. Thermostable DNA polymerase (Taq) - synthesises new strands of DNA
2. A pair of (specific) primers (forward and reverse) - uniquely define the region to be copied, starting point for DPol elongation
3. Free dNTPs - to build new DNA strands

Question 3

Describe the steps of PCR.

Answer 3

Consists of 20-40 temperature cycles, each involving 3 steps:

1. Denaturing (95 C)
   - Disruption of hydrogen bonds between complementary base pairs - forms ssDNA.
2. Annealing (50-65 C)
   - Annealing of primers to complementary DNA regions.
   - DPol binds to primer-template hybrid and begins polymerisation.
3. Polymerising (72 C)
   - DPol synthesises a complementary DNA strand in 5’-3’ direction.

Question 4

What are the applications of PCR?

Answer 4

• Restriction analysis
• Gene cloning
• DNA hybridisation and southern/northern blotting
• Reverse transcriptase-PCR

Question 5

What is RT-PCR?

Answer 5

PCR variant used to qualitatively detect gene expression through creation of complementary DNA (cDNA) transcript from RNA.

Question 6

Describe the process of RT-PCR.

Answer 6

1. A long T primer is made which binds to the mRNA poly A tail.
2. Reverse transcriptase then forms an RNA-cDNA duplex.
3. The RNA is broken down to have a ssDNA copy of the RNA.
4. This template DNA is amplified using normal PCR.

Question 7

Which techniques are used to analyse DNA at the nucleotide level?

Answer 7

1. Restriction analysis + gel electrophoresis

2. DNA sequencing

Question 8

Which enzymes are required to perform restriction analysis?

Answer 8

Restriction endonucleases = bacterially-produced enzymes which degrade foreign DNA by recognising and cutting specific DNA sequences (restriction sites) - mostly palindromes of 4, 5, 6 or 8 bp.

Question 9

What is the principle behind gel electrophoresis?

Answer 9

• DNA is negatively charged and will move towards the positive electrode (anode) if placed in an electric field.
• Smaller DNA fragments move faster than larger ones - DNA separated on the basis of size.

Question 10

What are the requirements for restriction analysis/gel electrophoresis?

Answer 10

1. Gel - matrix that allows DNA fragment separation
2. Buffer - allows charge on the DNA samples across the gel
3. Power supply - generates charge difference across the gel
4. Stain/detection - to identify presence of separated DNA (e.g. Ethium bromide)

Question 11

What are the applications of restriction analysis (+ PCR)?

Answer 11

• Investigate DNA fragment size, e.g. Small deletion
• Investigate mutations, e.g. Sickle cell disease
• Investigate DNA variation, e.g. DNA fingerprinting
• DNA cloning

Question 12

Which molecules does the Sanger chain termination method (dideoxy chain termination method) of DNA sequencing use?

Answer 12

• Uses modified nucleotides: dideoxynucleotide triphosphate (ddNTP) molecules - similar to typical dNTPs but contain H rather than OH at 3’ position.
• Means that although it can be used by DPol as a substrate, it will block further elongation if incorporated into a growing DNA strand - no further phosphodiester bonds will be formed with a subsequent dNTP.

Question 13

Describe the Sanger chain termination method (dideoxy chain termination method) of DNA sequencing.

Answer 13

1. 4 separate tubes are used, each with a unique ddNTP and a labelled primer to initiate DNA synthesis.
2. After incubation, reaction products undergo gel electrophoresis - separates labelled fragments out on basis of size.
3. Read out sequence from bottom of gel to work out nucleotide sequence in newly synthesised strand.

Question 14

How has the Sanger method of DNA sequencing been modernised?

Answer 14

• Now use fluorescently labelled ddNTPs all together in the same tube.
• Results can be produced as a chromatography from which we can read the sequence directly.

Question 15

Which methods can be used to analyse DNA at the gene level?

Answer 15

1. Gene cloning
2. DNA hybridisation + southern/northern blotting
3. RT-PCR
4. Microarrays
5. DNA fingerprinting/DNA profiling

Question 16

What is a plasmid?

Answer 16

• Small circular dsDNA found in bacteria.

- Carry genes to replicate independently, can transfer to other bacteria and often carry antibiotic resistance genes.

Question 17

Describe the process of gene cloning.

Answer 17

I. Isolate relevant gene following restriction digest or PCR.
II. Insert gene of interest into plasmid vector - forms recombinant DNA molecule.
III. Introduce recombinant DNA molecules into suitable host cells (e.g. E. coli).
IV. Isolate the clone containing the DNA of interest.

Question 18

Why clone human genes?

Answer 18

• Make useful proteins, e.g. Insulin
• Find out gene functions, e.g. HTT
• Genetic screening, e.g. Huntington’s, CF
• Gene therapy?, e.g. CF

Question 19

Describe the process of DNA hybridisation.

Answer 19

1. Denaturing: dsDNA is heated (or treated with alkaline solution), breaking H bonds and forming ssDNA.
2. Addition of ssDNA that is radioactively labelled.
3. Annealing: ssDNA is cooled - H bonds reform to make dsDNA. Some strands reanneal with labelled DNA.
4. Identify labelled DNA using photographic film.

Question 20

What are the 2 main types of hybridisation?

Answer 20

• Southern blotting: uses DNA probes to identify complementary DNA sequences after gel electrophoresis.
• Northern blotting: uses DNA to detect RNA species in a similar way to the above.

Question 21

Describe the process of southern blotting.

Answer 21

1. Production of DNA fragments (from genomic DNA/cloned gene/PCR) via restriction digest.
2. Gel electrophoresis to separate DNA fragments.
3. Transfer of DNA (denatures to ssDNA after soaking in alkaline solution) to nylon/nitrocellulose filter: blotted by capillary action or electrophoretic transfer.
4. Hybridisation of labelled DNA probe to specific piece of DNA (complementary sequence).
5. Visualisation of labelled probe using photographic film or fluorescent detection methods.

Question 22

What are the characteristics of the DNA probes used in southern blotting?

Answer 22

1. Do not have to have 100% complementarity to target sequence.
2. Do not have to completely align with target sequence.
3. Do not affect the position of the target sequence on a gel.

Question 23

What is southern hybridisation used to investigate?

Answer 23

• Gene structure, e.g. Large deletions/duplications
• Gene expansions, triplet repeats, e.g. Huntington’s
• Mutations in genetic tests using allele specific probes, e.g. Sickle cell disease
• Variation, genetic relationships, e.g. DNA fingerprinting

Question 24

What are microarrays and what do they investigate?

Answer 24

• Allows the analysis of 1000s of genes - ‘genome-wide’ analysis.
• Can be used to investigate:
  
  1. Conditional gene expression - which genes are switched on/off in 2 conditions (e.g. Disease vs health)
  2. Array comparative genome hybridisation - gene duplication/deletion

Question 25

Describe the process of microarrays investigating conditional gene expression.

Answer 25

1. RNA isolated from diseased cells and normal cells. 2. Use reverse transcriptase to label cDNA with fluorescent probes - red and green. 3. Combine arrest and hybridise to microarray. 4. Determine colour intensity in each representing gene 'spot'.

Question 26

Describe the process of microarray investigating gene duplication/deletion (array comparative genome hybridisation).

Answer 26

1. Extract DNA from diseased and normal cells. 2. Label with different fluorochromes (red and green). 3. Mix in equal quantities and hybridise to microarray of clones. 4. Work out red:green ration for each cell.

Question 27

What is DNA fingerprinting/DNA profiling?

Answer 27

- Doesn't look at genes but at non-coding regions (98% of genome) - contain many repeat areas 30-60 bp long (= mini-satellites). There are variable numbers of these repeats on homologous chromosomes at equivalent loci - determines locus size, causing allelic variation at each locus. - DNA fingerprinting uses these mini-satellites as probes - produce varying band patterns that show family relationships. - DNA profiling uses similar technique based on small tandem repeats, uses areas that are very variable in all humans.