Tools to generate data for Bioinformatics

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Tools to generate data for Bioinformatics

• Restriction enzyme digestion
• Restriction enzymes cleave ds DNA at specific
  sites, usually palindromic sequences
• 5 GAATTC 3
• 3 CTTAAG 5
• 5 G AATTC 3 (EcoRI)
• 3 CTTA G 5

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• The EcoRI digestion generated sticky ends
• The sticky ends can be covalently joined back
  together using DNA Ligase
• Restriction enzymes that do not create sticky
  ends can create blunt ends
• Example Hind II cleaves
• 5 - G T Py Pu A C 3
• 3 - C A Pu Py T G -5

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• Gel electrophoresis uses an agarose gel to
  separate DNA or RNA fragments according to size
• The DNA or RNA is blotted or transferred to a
  nylon membrane
• A radiolabeled DNA probe (usually 20 nts) is
  allowed to hybridize to DNA or RNA on the nylon
  membrane
• The nylon membrane is exposed overnight to x-ray
  film.

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• Restriction Fragment Length Polymorphism (RFLP)
• Restriction sites occur randomly in genomic with
  the probability of P (1/4)n where n number of
  nucleotides (usually 6 for restriction enzymes)
• So restriction site should occur about every
  4,096 nts

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• Sometimes a mutation occurs which destroys the
  restriction site.
• This results in a different pattern of DNA
  fragments from being generated than expected.
• We can use this unexpected pattern of DNA
  fragments to identify individuals.
• Thus RFLP can be used in forensic analysis

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MicroArray

• Short pieces of known DNA are arranged on a nylon
  membrane as a grid.
• mRNA from the target (i.e. tumor cells) is used
  as a template to synthesize cDNA
• The cDNA is synthesized using the reverse
  transcriptase
• The cDNA is labeled with flouresecent dyes and
  allowed to hybridize to the DNA on the nylon.
• The intensity of the resulting signal shows the
  relative abundance of the RNA

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• DNA fragments can be cloned into vectors (i.e.
  plasmids, lamda phage, Cosmids, Yeast Artificial
  Chromosomes (YACS)
• Genomic libraries can be constructed from the
  total genomic DNA inside cells.
• Genomic libraries usually use lambda phage,
  Cosmids and YACS
• cDNA libraries are constructed from cDNA
  synthesized from total mRNA templates

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• Traditional genomic and cDNA libraries allow only
  one sequence to be searched at a time.
• Hybridization of the probe to the target is only
  the first step. The target must be isolated and
  sequenced before analysis can begin.
• Low abundance targets in library may easily be
  missed by the probe.

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• Polymerase Chain Reaction (PCR)
• Highly sensitive technique for detecting low
  abundance DNA fragments
• Advantages quick (about 2 hours), inexpensive,
  requires little training
• Disadvantages only amplifies (copies) DNA
  fragments ( whose size less than 1.5 Kb), easily
  contaminated with previously PCR amplified DNA

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PCR steps

• DNA is heated to 92oC to denature the DNA
• DNA is cooled to 54oC to allow the DNA primers to
  anneal to the single stranded DNA
• Temperature is raised to 72oC to allow Taq
  polymerase (DNA polymerase) to synthesize new DNA
  strand
• Repeat previous steps 30 times

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