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PCR - Polymerase Chain Reaction - PowerPoint PPT Presentation

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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.

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Pcr polymerase chain reaction l.jpg
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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Primer selection l.jpg
Primer selectionhttp://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

  • 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.

Primer specificity l.jpg
Primer Specificityhttp://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

  • Universal – amplifies ALL bacterial DNA for instance

  • Group Specific – amplify all denitrifiers for instance

  • Specific – amplify just a given sequence

Forward and reverse primers l.jpg
Forward and reverse primershttp://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

  • If you know the sequence targeted for amplification, you know the size which the primers should be anealing across

  • If you don’t know the sequence… What do you get?

Dna polymerase l.jpg
DNA Polymerasehttp://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

  • 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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  • Restriction Fragment Length Polymorphism

  • Cutting a DNA sequence using restriction enzymes into pieces  specific enzymes cut specific places

Starting DNA sequence:



Enzyme X



Enzyme X









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  • 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 63:4516-4522

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  • 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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  • Denaturing gradient gel electrophoresis

    • The hydrogen bonds formed between complimentary base pairs, GC rich regions ‘melt’ (melting=strand 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/

Rflp vs dgge l.jpg

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


Very sensitive to variations in DNA sequence

Can excise and sequence DNA in bands


Somewhat difficult

”One band-one species” isn’t always true

Cannot compare bands between gels

Only works well with short fragments (<500 bp), thus limiting phylogenetic characterization



Relatively easy to do

Results can be banked for future comparisons


Less sensitive phylogenetic resolution than sequencing

Each fragment length can potentially represent a diversity of microorganisms

Cannot directly sequence restriction fragments,making identification indirect


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  • 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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Fluorescent in site hybridizationhttp://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

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B Drift Slime Streamerhttp://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

10 µm



Oligunucleotide design l.jpg
Oligunucleotide designhttp://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

Fish variations l.jpg
FISH variationshttp://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

  • 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

Clone library l.jpg
Clone Libraryhttp://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

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

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