1. 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.
5. 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.
6. Primer Specificity
• Universal – amplifies ALL bacterial DNA for instance
• Group Specific – amplify all denitrifiers for instance
• Specific – amplify just a given sequence
7. 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 don’t know the sequence… What do you get?
8. 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
9. RFLP
• Restriction Fragment Length Polymorphism
• Cutting a DNA sequence using restriction enzymes into pieces
specific enzymes cut specific places
Starting DNA sequence:
5’-TAATTTCCGTTAGTTCAAGCGTTAGGACC
3’-ATTAAAGGCAATCAAGTTCGCAATAATGG
Enzyme X
5’-TTC-
3”-AAG-
Enzyme X
5’-TTC-
3”-AAG-
5’-TAATTT
3’-ATTAAA
5’-CCGTTAGTT
3’-GGCAATCAA
5’-CAAGCGTTAGGACC
3’-GTTCGCAATAATGG
10. 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 63:4516-4522
11. Electrophoresis
• Fragmentation products of differing length are separated –
often on an agarose gel bed by electrophoresis, or using a
capilarry electrophoretic separation
12.
13. DGGE
• 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/
14. RFLP vs. DGGE
DGGE
• Advantages
– Very sensitive to variations in DNA
sequence
– Can excise and sequence DNA in bands
• Limitations
– 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
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
15. 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
16. 16S gene
16S rRNA
CellCell
membranemembrane
DNA
16S gene
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Fluorescent in situ hybridisation
(FISH) using DNA probes
TAGCTGGCAGT
AUCGACCGUCACGU
Fluorescein
AU
ProbeProbe
(( 20 bases)20 bases)
Fluorescent in site hybridization
20. 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