Polymerase Chain Reaction (PCR) is a technique that amplifies specific DNA sequences using oligonucleotide primers and DNA polymerase, crucial for laboratory DNA analysis. It is often paired with methods like Restriction Fragment Length Polymorphism (RFLP) and Denaturing Gradient Gel Electrophoresis (DGGE) for DNA separation and characterization. Fluorescent in-situ hybridization (FISH) is another method to image specific organisms in samples using tagged oligonucleotide probes.

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.

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

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

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
Cell
Cell
membrane
membrane
DNA
16S gene
*
*
*
*
*
*
*
*
*
* *
* *
*
* * *
Fluorescentin situhybridisation
(FISH) using DNA probes
TAGCTGGCAGT
AUCGACCGUCA
CGU
Fluorescein
A
U
Probe
Probe
(
(-
- 20 bases)
20 bases)
Fluorescent in site hybridization

17. 10 µm
DAPI FER656
B Drift Slime Streamer

19. Oligunucleotide design

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

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

22. http://www.ifa.hawaii.edu/UHNAI/NAIweb/presentations/astrobiol6.pdf