PCR - Polymerase Chain Reaction

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PCR - Polymerase Chain Reaction

• PCR is an in vitro technique for the
  amplification of a region of DNA which lies
  between two regions of known sequence.
• PCR amplification is achieved by using
  oligonucleotide primers.
• These are typically short, single stranded
  oligonucleotides which are complementary to the
  outer regions of known sequence.
• The oligonucleotides serve as primers for DNA
  polymerase and the denatured strands of the large
  DNA fragment serves as the template.
• This results in the synthesis of new DNA strands
  which are complementary to the parent template
  strands.
• These new strands have defined 5' ends (the 5'
  ends of the oligonucleotide primers), whereas the
  3' ends are potentially ambiguous in length.

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• http//ocw.mit.edu/NR/rdonlyres/Civil-and-Environm
  ental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377
  -8D74-8EEE753A328C/0/11_02_04.pdf

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Primer selection

• Primer is an oligonucleotide sequence will
  target a specific sequence of opposite base
  pairing (A-T, G-C only) of single-stranded
  nucleic acids
• For example, there is a
• ¼ chance (4-1) of finding an A, G, C or T in any
  given DNA sequence there is a
• 1/16 chance (4-2) of finding any dinucleotide
  sequence (eg. AG) a
• 1/256 chance of finding a given 4-base sequence.
• Thus, a sixteen base sequence will statistically
  be present only once in every 416 bases (4 294
  967 296, or 4 billion) this is about the size of
  the human or maize genome, and 1000x greater than
  the genome size of E. coli.

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Primer Specificity

• Universal amplifies ALL bacterial DNA for
  instance
• Group Specific amplify all denitrifiers for
  instance
• Specific amplify just a given sequence

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Forward and reverse primers

• If you know the sequence targeted for
  amplification, you know the size which the
  primers should be anealing across
• If you dont know the sequence What do you
  get?

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DNA Polymerase

• DNA Polymerase is the enzyme responsible for
  copying the sequence starting at the primer from
  the single DNA strand
• Commonly use Taq, an enzyme from the
  hyperthermophilic organisms Thermus aquaticus,
  isolated first at a thermal spring in Yellowstone
  National Park
• This enzyme is heat-tolerant ? useful both
  because it is thermally tolerant (survives the
  melting T of DNA denaturation) which also means
  the process is more specific, higher temps result
  in less mismatch more specific replication

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RFLP

• Restriction Fragment Length Polymorphism
• Cutting a DNA sequence using restriction enzymes
  into pieces ? specific enzymes cut specific places

Starting DNA sequence 5-TAATTTCCGTTAGTTCAAGCGTTA
GGACC 3-ATTAAAGGCAATCAAGTTCGCAATAATGG
Enzyme X 5-TTC- 3-AAG-
Enzyme X 5-TTC- 3-AAG-
5-CAAGCGTTAGGACC 3-GTTCGCAATAATGG
5-TAATTT 3-ATTAAA
5-CCGTTAGTT 3-GGCAATCAA
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RFLP

• DNA can be processed by RFLP either directly (if
  you can get enough DNA from an environment) or
  from PCR product
• T-RFLP (terminal-RFLP) is in most respects
  identical except for a marker on the end of the
  enzyme
• Works as fingerprinting technique because
  different organisms with different DNA sequences
  will have different lengths of DNA between
  identical units targeted by the restriction
  enzymes
• specificity can again be manipulated with PCR
  primers

Liu et al. (1997) Appl Environ Microbiol
634516-4522
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Electrophoresis

• Fragmentation products of differing length are
  separated often on an agarose gel bed by
  electrophoresis, or using a capilarry
  electrophoretic separation

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DGGE

• Denaturing gradient gel electrophoresis
• The hydrogen bonds formed between complimentary
  base pairs, GC rich regions melt
  (meltingstrand separation or denaturation) at
  higher temperatures than regions that are AT
  rich.
• When DNA separated by electrophoresis through a
  gradient of increasing chemical denaturant
  (usually formamide and urea), the mobility of the
  molecule is retarded at the concentration at
  which the DNA strands of low melt domain
  dissociate.
• The branched structure of the single stranded
  moiety of the molecule becomes entangled in the
  gel matrix and no further movement occurs.
• Complete strand separation is prevented by the
  presence of a high melting domain, which is
  usually artificially created at one end of the
  molecule by incorporation of a GC clamp. This is
  accomplished during PCR amplification using a PCR
  primer with a 5' tail consisting of a sequence of
  40 GC.

Run DGGE animation here from http//www.charite.
de/bioinf/tgge/
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RFLP vs. DGGE

• RFLP
• Advantages
• Relatively easy to do
• Results can be banked for future comparisons
• Limitations
• Less sensitive phylogenetic resolution than
  sequencing
• Each fragment length can potentially represent a
  diversity of microorganisms
• Cannot directly sequence restriction
  fragments,making identification indirect

• DGGE
• Advantages
• Very sensitive to variations in DNA sequence
• Can excise and sequence DNA in bands
• Limitations
• Somewhat difficult
• One band-one species isnt always true
• Cannot compare bands between gels
• Only works well with short fragments (lt500 bp),
  thus limiting phylogenetic characterization

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FISH

• Fluorescent in-situ hybridization
• Design a probe consisting of an oligonucleotide
  sequence and a tag
• Degree of specificity is variable!
• Hybridize that oligonucleotide sequence to the
  rRNA of an organism this is temperature and
  salt content sensitive
• Image using epiflourescence, laser excitation
  confocal microscopy
• Technique DIRECTLY images active organisms in a
  sample

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B Drift Slime Streamer
10 µm
DAPI
FER656
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Oligunucleotide design
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FISH variations

• FISH-CARD instead of a fluorescent probe on
  oligo sequence, but another molecule that can
  then bond to many fluorescent probes better
  signal-to-noise ratio
• FISH-RING design of oligo sequence to specific
  genes image all organisms with DSR gene or nifH
  for example

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Clone Library

• http//ocw.mit.edu/NR/rdonlyres/Civil-and-Environm
  ental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377
  -8D74-8EEE753A328C/0/11_02_04.pdf

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http//www.ifa.hawaii.edu/UHNAI/NAIweb/presentatio
ns/astrobiol6.pdf
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