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Lecture 3

The document summarizes techniques used in PCR (polymerase chain reaction) amplification and analysis of DNA sequences. PCR is used to amplify a targeted region of DNA between two known primer sequences. The primers and DNA polymerase are used to synthesize new DNA strands that are complementary to the template strands. The new DNA fragments have defined 5' ends from the primers. Various techniques can then analyze the amplified DNA fragments, including restriction fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE), fluorescent in situ hybridization (FISH), and clone libraries.

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113 views

Lecture 3

The document summarizes techniques used in PCR (polymerase chain reaction) amplification and analysis of DNA sequences. PCR is used to amplify a targeted region of DNA between two known primer sequences. The primers and DNA polymerase are used to synthesize new DNA strands that are complementary to the template strands. The new DNA fragments have defined 5' ends from the primers. Various techniques can then analyze the amplified DNA fragments, including restriction fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE), fluorescent in situ hybridization (FISH), and clone libraries.

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nguyen ba trung
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© © All Rights Reserved
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Download as PPT, PDF, TXT or read online on Scribd
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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.
• http://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf
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.
Primer Specificity
• Universal – amplifies ALL bacterial DNA
for instance
• Group Specific – amplify all denitrifiers for
instance
• Specific – amplify just a given sequence
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?
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
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 Enzyme X
5’-TTC- 5’-TTC-
3”-AAG- 3”-AAG-

5’-TAATTT 5’-CCGTTAGTT 5’-CAAGCGTTAGGACC


3’-ATTAAA 3’-GGCAATCAA 3’-GTTCGCAATAATGG
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


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


RFLP vs. DGGE
RFLP DGGE
• Advantages • Advantages
– Relatively easy to do – Very sensitive to variations in
– Results can be banked for DNA sequence
future comparisons – Can excise and sequence
• Limitations DNA in bands
– Less sensitive phylogenetic • Limitations
resolution than sequencing – Somewhat difficult
– Each fragment length can – ”One band-one species” isn’t
potentially represent a diversity always true
of microorganisms – Cannot compare bands
– Cannot directly sequence between gels
restriction fragments,making – Only works well with short
identification indirect fragments (<500 bp), thus
limiting phylogenetic
characterization
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
Fluorescentininsitu
Fluorescent sitehybridisation
hybridization
(FISH) using DNA probes
Probe Fluorescein
(- 20 bases) TA GC TG G C A G T
C G UAUCGAC C G UC A
UA
16S rRNA
DNA *
16S gene
* * * *
*
* *

*
*

*
*
* *
16S gene * * Cell
membrane
B Drift Slime Streamer
10 µm

DAPI FER656
Oligunucleotide design
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
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
http://www.ifa.hawaii.edu/UHNAI/NAIweb/presentations/astrobiol6.pdf

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