Recombinant DNA:

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Chapter 17

• Recombinant DNA
• The Tools of the Trade

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Restriction enzymes

• Discovered in the 1970s
• Used by bacteria to break up viral DNA
• Bacteria DNA is preserved by being methylated
• Reads in paladromes
• The DNA fragment produced by resptriction enzymes
  acting on the DNA of a cell are call genomicDNA
  gDNA

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• Animation of the ligation process. When the
  sticky ends of two DNA fragments come together
  and hydrogen bond through the A-T and G-C
  pairing, the enzyme LIGASE will form covalent
  bonds between the two fragments.

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• Once you have the DNA you want you will want to
  make many copies of it.
• PCR (Polymerase Chain Reaction) Nobel Prize 1993
• AMPLIFYING our DNA millions of time.

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PCR The components are

• A sample of the TARGET DNA to be copied. In
  theory only a single molecule is needed.
• A set of short (15 to 40 bases) single stranded
  PRIMERS of DNA, in EXCESS, that will bind to
  complementary regions of the opposing stands of
  the TARGET DNA molecule . These primers BRACKET
  the region of DNA to be amplified.
• An EXCESS of the 4 nucleotide triphosphates,
  ATP, GTP, CTP, TTP.
• The enzyme, DNA polymerase.
• Various buffers and cofactors like magnesium
  ions required by DNA polymerase.

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• The final trick was to get the two target DNA
  strands APART (separated) so the primers could
  bind and the DNA polymerase could do its thing
• Mullis heated his mixture of target DNA, primers
  and triphosphate nucleotides to about 90oC for a
  few minutes to separate the target DNA.
• then lowered the temperature enough to allow the
  primers, which were small and in VAST EXCESS, to
  bind (ANNEAL) to their respective complementary
  target DNA base pair-sequences.
• At this point he added DNA polymerase and allowed
  the polymerization reaction with the triphosphate
  nucleotides to occur.
• That is, the DNA polymerase FILLED IN the missing
  portion of each strand making TWO NEW DOUBLE
  STRANDED regions of DNA

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Reverse Transcriptase cDNAcomplementary DNA

• Bacteria is used to make large quanities of
  eukaryotic genes and protiens
• Eukaryote have introns and exons
• Prokaryotes do not
• The trick is to modify the gene you want into a
  gene the prokaryotes can transcibe
• Reverse transcriptase

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Synthetic Oligonucleotides

• Selectively block either the 3 end or the 5 end
• Ensures that the condensation reactions forming
  the sugar phosphate bond occur in the proper
  sequence for the desired order

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Antisense oligonucleotides are synthetic
fragments of ribo or deoxyribonucleic acids which
specifically binds to their complementary
messenger RNA and then, block the corresponding
protein translation. 
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Plasmids as Vector

• Restriction enzymes are used
• Ex
• EcoRI
• Useful as vectors b/c multiply rapidly and are
  easily taken up by bacteria through cell membrane
• Multiple copies are called clone

Use of plamids Insulin and Human GH
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Radioactive Probes

• Used to isolate DNA or mRNA
• To do this application of nucleic acid
  hybridzation, using radioactive probes
• Probes short segment of single-stranded DNA or
  RNA labeled with a radioactive isotope

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DNA Sequencing
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