Molecular Techniques - DNA Flashcards

1
Q

Which technique is used to amplify DNA?

A

Polymerase Chain Reaction

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2
Q

What components are required for PCR?

A
  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
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3
Q

Describe the steps of PCR.

A

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.
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4
Q

What are the applications of PCR?

A
  • Restriction analysis
  • Gene cloning
  • DNA hybridisation and southern/northern blotting
  • Reverse transcriptase-PCR
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5
Q

What is RT-PCR?

A

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

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6
Q

Describe the process of RT-PCR.

A
  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.
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7
Q

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

A
  1. Restriction analysis + gel electrophoresis

2. DNA sequencing

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8
Q

Which enzymes are required to perform restriction analysis?

A

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.

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9
Q

What is the principle behind gel electrophoresis?

A
  • 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.
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10
Q

What are the requirements for restriction analysis/gel electrophoresis?

A
  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)
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11
Q

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

A
  • Investigate DNA fragment size, e.g. Small deletion
  • Investigate mutations, e.g. Sickle cell disease
  • Investigate DNA variation, e.g. DNA fingerprinting
  • DNA cloning
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12
Q

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

A
  • 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.
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13
Q

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

A
  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.
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14
Q

How has the Sanger method of DNA sequencing been modernised?

A
  • 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.
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15
Q

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

A
  1. Gene cloning
  2. DNA hybridisation + southern/northern blotting
  3. RT-PCR
  4. Microarrays
  5. DNA fingerprinting/DNA profiling
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16
Q

What is a plasmid?

A
  • Small circular dsDNA found in bacteria.

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

17
Q

Describe the process of gene cloning.

A

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.

18
Q

Why clone human genes?

A
  • 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
19
Q

Describe the process of DNA hybridisation.

A
  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.
20
Q

What are the 2 main types of hybridisation?

A
  • 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.
21
Q

Describe the process of southern blotting.

A
  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.
22
Q

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

A
  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.
23
Q

What is southern hybridisation used to investigate?

A
  • 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
24
Q

What are microarrays and what do they investigate?

A
  • 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
25
Q

Describe the process of microarrays investigating conditional gene expression.

A
  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’.
26
Q

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

A
  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.
27
Q

What is DNA fingerprinting/DNA profiling?

A
  • 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.