Molecular Biology Techniques Flashcards

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1
Q

Polymerase Chain Reaction

A

95 – denaturation of ds DNA by the breaking of hydrogen bonds between complementary base pairs due to increased molecular vibrations due to high heat

64 – primer annealing
- each primer anneals specifically to the. 3’ end of each single stranded target DNA sequence via complementary base pairing when. the temperature is lowered

72 – Taq polymerase synthesises a complementary DNA strand from the free 3’OH end of the DNA primer by catalysing the formation of phosphodiester bonds between dNTPS

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2
Q

advantages of PCR

A
  • only a minute amount of DNA is required to carry out PCR with each round of PCR, the number of copies of target DNA is doubled –>. number of desired sequence increases exponentially and there will be sufficient DNA for analysis
  • use of thermostable Taq polymerase allows PCR to be automated so DNA can be amplified very quickly
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3
Q

limitations of PCR

A
  • Taq polymerase lacks 3’ to 5’ proofreading ability, hence errors occurring early in the PCR reaction will get compounded with subsequent replication cycle
  • knowledge of sequences flanking the target sequence is required in order to design appropriate primers
  • Taq pol tends to fall off the DNA template before chain extension is complete if the strand is too long, there is a limit to the size of DNA fragment
  • minute amounts of contaminant DNA can be exponentially amplified along with the target DNA and affect reliability of results
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4
Q

Principles and procedures of gel electrophoresis

A
  • separate DNA based on fragment size
  • DNA sample is mixed with dense loading dye to make DNA sample denser to sink to the bottom of the well
  • negatively charged DNA migrates from the negative electrode to the positive electrode
  • meshwork of agarose gel fibres impede movement of longer fragments more than that of shorter fragments, hence longer fragments migrate slower and end up nearer to the well
  • to visualise the bands, the gel can be treated with a staining dye (ethidium bromide) that binds DNA and fluoresces under UV light
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5
Q

Principles and procedures of Southern Blotting

A
  • detect specific nucleotide sequences within a sample of DNA
  • gel slab is placed under a nitrocellulose membrane, placed in alkaline solution to denature double stranded DNA into ss DNA
  • ss DNA is drawn upwards onto the nitrocellulose membrane and binds to the membrane.
  • incubated with ss radioactive DNA probes which hybridises via complementary base pairing to part if the target sequence
  • autoradiography is performed placing X ray film over membrane
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6
Q

Restriction Fragment Length Polymorphism

A
  • unique banding patterns among individuals when their DNA is digested by restricted enzymes and separated by gel electrophoresis.
  • RFLPs arise due to polymorphic nature of DNA, there will be variations in number and location of restriction sites and number of tandemly repeated sequences
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7
Q
A
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