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• 1 DNA replication, sequencing and PCR

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• Why is DNA replication considered semi-conservative?

  Because every new double stranded molecule consist of one old and one new strand
  

  • the old strand is the template 

• What is the chemical reaction behind DNA synthesis?

  The formation of phosphodiester bonds while hydrolysing the matching dNTP molecule of the template strand

• What are the frequency of mistakes?

  1. Without proof-reading: 1 in 10^5
     
  2. with proof-reading: 1 in 10^7
  3. with strand-directed mismatch repair: 1 in 10^10

• What is strand-directed mismatch repair?

  When there is a error in the new strand, mismatch proofreading proteins will bind to DNA and detects error in strand. The error will be removed and the DNA strand will be repaired.

• What causes permanent changes in the template strand?

  Chemical changes in DNA bases can cause mutation in the DNA
  

  • depurination: loss of purine bases (A and G), will cause deletion in mutated strand
  • deamination: C -> U

• What are the main differences between DNA sequencing and PCR?

  DNA sequencing determine the order of the bases, while PCR amply the DNA

• What is shotgun sequencing?

  The method involves randomly breaking up the genome into small DNA fragments that are sequenced individually. A computer program looks for overlaps in the DNA sequences, using them to reassemble the fragments in their correct order to reconstitute the genome.
  

  • works well for small genomes that lack repetitive DNA

• What are the difference between different sequencing platform?

  Read length and error rate

• What are the advantages and restrictions of PCR?

  Advantages: sensitive and fast
  Restrictions: fragment length <20kb and homologous DNA sequences

• What are the techniques for detecting DNA polymorphism?

  PCR-based: VNTR
  PCR and sequencing-based/melting curve: SNP