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.

• http://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 .

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

• 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 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

• 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

• 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

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

• 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
• 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” isn’t always true
– Cannot compare bands between gels
– Only works well with short fragments (<500 bp), thus limiting phylogenetic characterization

• 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

DNA
Fluorescein
( ( 20 bases)
16S rRNA
*
C GU
TAGCTGGCAGT
AUCGACC GUC A
U A
*
*
16S gene
*
*
*
* * *
*
*
*
*
* *
16S gene *
*

10 µm
B Drift Slime Streamer
DAPI FER656

• 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

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

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