mbt

• polymerase chain reaction:

  1. heating to 95 degrees denatures ds dna to ss dna as heat breaks weak H bonds
  2. in the presence of large excess of dna primers, each dna primer anneals specifically to 3'oh end of ss target dna via cbp, when T is lowered to 64 degrees
  3. taq polymerase synthesises complementary dna strand from 3'oh end of dna primer by catalysing formation of phosphodiester bonds between dNTPs when T is raised to 72 degrees
• n cycles of pcr synthesises 2^n ds dna
• why is 25-30 rounds of pcr reccomended?

  too few: not enough dna for analysis

  too many: too many mistakes in rep
• adv of pcr:

  1. speed: use of thermostable taq polymerase allows pcr to be automated so dna can be amplified fast and efficiently
  2. sensitivity: only a minute amt of dna req for pcr, as with each round of pcr, no. of copies of dna is doubled
• limitations of pcr:

  1. taq polymerase lacks 3' to 5' proofreading ability, errors occuring early in pcr reaction will get compounded with each subsequent replication cycle
  2. requires sequence information of target region for synthesis of appropriate primers
  3. dna fragment limited to maximum size of about 3kb, taq polymerase tends to fall off dna template before chain extension is complete
  4. contaminated dna can be exponentially amplified along with target sequence, affecting reliability of results
• process of mbt:

  1. extract dna sample
  2. pcr
  3. cut dna using restriction enzyme
  4. gel electrophoresis
  5. visualise dna

  • using ethidium bromide + uv light
  • southern blotting
• • a stab of agarose gel is placed in a buffer soln containing ions that allow electricity conduction when current turned on
  • dna sample mixed with dense loading dye , containing glycerol and 2 loading dyes. glycerol makes dna sample denser than buffer soln so dna can sink to bottom of well
  • as dna is invisible, the 2 loading dyes monitor the process of GE. dark blue dye corresponds to 100 bp frag, light blue 1100 bp frag
  • a dna ladder containing mixture of dna frags of known sizes, act as standards to be compared with frags of unknown sizes in sample
• • when current turned on, all dna fragments migrate out of well, into agarose. -ve charged dna is attracted towards anode.
  • agarose gel is made of meshwork of polymer fibres, impeding movement of longer dna frag more than shorter ones
  • current is turned off before loading dye reached end of gel
• restriction enz:

  • recognize & bind to specific palindromic base sequences on DNA
  • create either sticky ends/blunt ends by cleaving of phosphodiester bonds between nucleotides in both strands of DNA
  • protect bacterial cells from invading viruses by degrading the foreign DNA that enter them
  • do not cleave the bacterial DNA in the bacteria they are found in as the restriction sites in the bacterial DNA are methylated. Methylation protects
  • bacterial DNA from degradation.
• 5a. visualise dna with ethidium bromide + uv light

  • stain gel w EB and fluorensce under UV light
  • fragment size (position of band relative to position of bands in dna ladder) & amt of dna (thickness of band) can thus be estimated
• 5b: visualise dna via southern blotting

  • gel slab placed on top of a sponge and under a nitrocellulose membrane. a stack of paper towels are placed on top of NM. these are placed in an alkaline solution. a heavy weight is then placed on paper towels
• 5b: southern blotting

  • absorbent paper towels draw solution towards themselves and the alkaline solution denatures ds dna to ss dna
  • ss dna is then drawn upwards into NM and binds to membrane in exactly same position as they were in the gel
  • NM is removed and incubated with ss radioactive dna probes which hybridise via cbp to target sequence,. excess unhybridised probes are washed off
  • autoradiography is performed by placing X ray film over membrane, radioactive regions exposes film, forming an image that corresponds to bands that have hybridised to probe
• RFLP

  • due to diff alleles producing diff bands in gel, resulting in unique banding patterns in individuals
  • diff bands arise from polymorphic nature of dna in homologpus regions in diff indiv, resulting in variation in number and location of restriction sites & number of tandem repeat sequences among individuals
  • genetic fingerprinting of X can be compared to Y to see how closely related they are
  • if fingerprint patterns are similar...
• how can RFLP facilitate GF

  • if genomes of 2 indiv cut with same restiction enzyme
  • if 2 indic differ in no. of tandem repeats at a particular locus at a particular chromosome
  • indiv with larger no. of tandem repeats generate longer restriction fragments which travel a shorter distance down gel during GE, vice versa
  • 2 different sized fragments are detected using same probe which has a sequence complementary to segments of both fragments
  • discriminating enough to gen unique genetic profile for each indiv
• why should cut at X

  RE should not cut within X as will result in same sized RF, unable to distinguish btwn different alleles
• outline principle of GE

  1.when subjected to current, neg charged dna migrates out of well towards anode

  2.meshwork of polymer fibre that make up agarose gel impedes movement of dna fragments

  3.longer dna fragments migrate slower...
• purpose of control for pcr

  if bands of expected size is not seen on gel, reagents not working properly
• why comparing more loci is better

  when using single locus, possibility of 2 indiv having same banding pattern, esp if they are related

  using more will give rise to unique banding pattern

  probability of 2 indiv w identical banding pattern for all 13 loci is v low

  able to distinguish btwn indiv, allow accurate identification
• primer role in pcr

  provides free 3'oh for taq polym for attachment and start chain extension
• why are 2 different primers needed

  to recog & bong to 2 diff 3' end as dna is ds, dna seq at 3' ends different