Advanced Molecular Biological Techniques

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Advanced Molecular Biological Techniques
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Polymerase chain reaction(PCR)

• Creates DNA sequence by repeated cycles of strand
  separation and replication
• In DNA replication DNA gyrase and helicase
  separate the DNA strands, in PCR heat separates
  the 2 (94-96 degrees Celsius) H-bonds separate
  in high temp

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Polymerase chain reaction(PCR)

• In replication DNA polymerase III is used to
  create complimentary strands, it requires an RNA
  primer to start, works in 5-3 direction
• In PCR DNA primers replace RNA primers these are
  easy to synthesize in the labs
• 2 primers one compliments one end and the other
  compliments the other end of the DNA, one is a
  forward primer and one is a reverse (one builds
  5-3, the other builds 3-5)

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Polymerase chain reaction(PCR)

• temperature is brought down to 50 65 degrees
  Celsius for primers to anneal with template DNA
• after they anneal, Taq polymerase can build
  complimentary strands
• synthesis takes place at 72 degrees Celsius

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Polymerase chain reaction(PCR)

• Taq polymerase is taken from Thermus Aquaticus, a
  bacterium that lives in hot springs
• Why is this important? Their enzymes can
  function at high temperatures
• DNA polymerase denatures above 37 degrees Celsius
  explains why a fever is so bad
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Restriction Fragment Length Polymorphism

• RFLP
• polymorphism any difference in DNA sequencing,
  coding or non-coding, that can be detected
  between individuals
• organisms of the same species have same genes but
  different alleles
• identical twins do not have polymorphism
• occur both in coding and non-coding regions

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RFLP

• restriction fragment length polymorphism (RFLP)
  analysis a technique in which DNA regions are
  digested using restriction endonuclease(s) and
  subjected to radioactive complementary DNA probes
  to compare the differences in DNA fragment
  lengths between individuals

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RFLP

• the DNA is first digested using one or more
  restriction endonucleases
• Different individuals have restriction sites at
  different locations. Each person will have
  different lengths of fragments (polymorphism)
• differences in patterns of fragments must be
  detected to distinguish DNA from 2 different
  sources

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RFLP

• Southern Blotting
• a chemical that causes double stranded DNA to
  split into single stranded DNA is introduced into
  the gel
• Single stranded DNA is transferred to a nylon
  membrane with an electric current
• The nylon mb is dipped into a solution with
  radioactive nucleotides

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RFLP

• Hybridization complementary base pairing
  between strands of nucleic acids via hydrogen
  bonding
• Nylon mb is then placed against an X-ray film to
  see their locations - known as autoradiogram
• You can use this to match the pattern of DNA in a
  crime scene blood at the scene of the crime and
  the suspects

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RFLP

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DNA sequencing

• Sanger dideoxy method DNA sequencing technique
  based on DNA replication that uses dideoxy
  nucleoside triphosphates
• This methods uses the process of DNA replication
• Replication requires a single stranded template,
  a primer, DNA polymerase, nucleoside
  triphosphates
• In the Sanger method the DNA template is treated
  to become single stranded DNA.

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DNA sequencing

• A short, single stranded, radioactive primer is
  added to one end
• Identical copies of the primed single stranded
  DNA are put into 4 reaction tubes, each tube has
  polymerase and free nucleotides for building

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DNA sequencing

• Each of the 4 tubes has a different radioactively
  labeled dideoxy analogue
• This is a nucleoside triphosphate whose ribose
  sugar does not possess a hydroxyl group on the 2
  and the 3 carbon
• There is a dideoxy analogue for each base pair
• When a dideoxy analogue is incorporated it stops
  the chain from growing
• This method is also known as a termination
  technique

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DNA sequencing

• If a desired strand to be copied is 3
  AATGCATGCATTAGC 5 and this is in a solution with
  dideoxy analogue adenine then the sequence will
  potentially stop where ever there is the base A.
• Dideoxy will stop the chain from growing.
• Different lengths will be separated by gel
  electrophoresis

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DNA sequencing

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