Chapter 13 - Molecular Methods
Objectives
1)
2)
3)
Be able to describe what a gene probe is and what it can be used for.
Understand the PCR reaction.
Be able to describe the different types of PCR: normal, RT-PCR, ICC-PCR,
multiplex PCR, seminested PCR, PCR fingerprinting, real-time PCR, in situ
PCR. Be able to give an example of the use of each of these types of PCR.
4) Understand the different types of PCR fingerprinting techniques including
AP-PCR, REP-PCR, ERIC-PCR. Be able to give an example application of
a PCR fingerprinting technique.
5) Understand RFLP and its application to forensics.
6) Be able to define cloning, cloning vector, and alpha-complementation.
7) Understand the concept of metagenomic analysis
8) Understand DGGE and TRFLP analysis and its use in community analysis.
9) Be able to define what a reporter gene is and know the different types of
reporter genes. Be able to give an example of how each of the different
types reporter genes is used.
10) Be able to define what a microarray is and to give an example of how a
microarray could be used to monitor a microbial community.
Molecular techniques are based on the structure of DNA and RNA
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Gene probes
A gene probe is a short specific sequence of DNA that is used to query
whether a sample contains “target” DNA, or DNA complementary to the
gene probe.
Gene probe (usually 100-500 bp in length)
Single strand of DNA
ACCGTAAT
CCTAAAGTGGCATTACCCTTGAGCTA
Target sequence
The target sequence can be a universally conserved region such as the
16S-rDNA gene or it can be in a region that is conserved within a specific
genus or species such as the nod genes for nitrogen fixation by Rhizobium
or the rhl genes for rhamnolipid biosurfactant production by Pseudomonas
aeruginosa.
PCR-Polymerase Chain Reaction
In many cases there is not enough DNA in a sample for a gene probe to
detect. Sample DNA can be amplified using PCR.
Need:
• Target DNA
• Primers: 17 to 30bp, GC content >50%
• Primers can be for universal conserved sequences (16S rDNA,
dehydrogenase genes) or genus-level conserved sequences (Nod,
Rhl, LamB genes)
• dNTPs
• DNA polymerase (original was taq polymerase from Thermus
aquaticus. Now there are several other DNA polymerases available)
PCR Round 1
target DNA
5'
3'
3'
5'
5'
3'
3'
Denaturation
5'
5'
3'
3'
Primer annealing
5'
5' 5'
3' 3'
Double-stranded DNA
3' 3'
5' 5'
5' 5'
3' 3'
repeat PCR cycles
3' 3'
5' 5'
Extension
DNA polymerase always adds nucleotides to
the 3’ end of the primer
5'
PCR Round 2
3'
5'
3'
3'
5'
5'
3'
After the second round of
PCR, the number of long
strands increases
arithmetically and the
number of short strands
increases exponentially
(the number of
chromosomal strands is
always the same).
5'
3'
5'
3'
denaturation
5'
3'
3'
5'
5'
3'
3'
5'
primer annealing
3'
5'
5'
3'
5'
3'
5'
3'
3'
5'
5'
3'
Short strand
3'
5'
5'
3'
Chromosomal strand
3'
5'
Long strand
extension
Temperature control in a PCR thermocycler
Temperature 0C
94 0C - denaturation
50 – 70 0C - primer annealing
72 0C - primer extension
94 0C - denaturation
After 25 cycles have 3.4 x 107 times more DNA
plateau is reached after
25-30 cycles
# PCR cycles
A PCR product should be confirmed in at least two ways initially.
These can include:
1. Correct product size.
2. Sequence the product.
3. Use a gene probe to confirm the product.
4. Use seminested PCR (see later)
RT-PCR
The enzyme reverse transcriptase is used to make a DNA copy (cDNA)
of an RNA template from a virus or from mRNA.
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Normal PCR with two primers
Multiplex PCR
Use of multiple sets of primers to detect more than one organism or to
detect multiple genes in one organism. Remember, the PCR reaction is
inherently biased depending on the G+C content of the target and primer
DNA. So performing multiplex PCR can be tricky.
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Seminested PCR
Three primers are required, the normal upstream and downstream primers as
well as a third, internal primer. Two rounds of PCR are performed, a normal
PCR with the upstream and downstream primer, and then a second round of
PCR with the downstream and internal primer. A second smaller product is
the result of the second round of PCR.
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ICC-PCR
Integrated cell culture PCR is used for virus detection. Cell culture takes 10 – 15
days. PCR alone detects both infectious and noninfectious particles. So use a
combination of these techniques: grow the sample in cell culture 2 – 3 days,
release virus from cells and perform PCR. This results in the detection of
infectious virus in a shorter time with a 50% cost savings. It also allows use of
dilute samples which reduces PCR inhibitory substances.
Real-Time PCR
This technique allows quantitation of
DNA and RNA. Reactions are
characterized by the point in time
during cycling when amplification of a
PCR product is first detected rather
than the amount of PCR product
accumulated after a fixed number of
cycles. The higher the starting copy
number of the nucleic acid target, the
sooner a significant increase in
fluorescence is observed.
Labelling approaches
SCYBR
Y B R G r eengreen
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ssD N A -- unbound dye
m inim a l fluoresce nce
dsD N A -- bound dye
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T aq M an -- H yd rolysis
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FRET
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M onitor acce ptor fluorescence
Figure 2. Figure X. Schematic of SYBR Green I, TaqMan, and hybridization probe
PCR fingerprinting
AP-PCR (arbitrarily primed PCR), 1 primer required, 10-20 bp, no
sequence information required
REP-PCR (repetitive extragenic palindromic sequences) 2 primers
insert randomly into the REP sites
ERIC-PCR (enterobacterial repetitive intergenic consensus sequences),
2 primers insert randomly into the ERIC sites, best for Gram Negative
microbes
All of these fingerprinting techniques tell one if two isolates are the same
or different. They do not provide information about the identity or
relatedness of the organisms
RFLP Fingerprinting Analysis
RFLP = restriction fragment length polymorphism
RFLP analysis involves cutting DNA into fragments using one or a set of
restriction enzymes.
For chromosomal DNA the RFLP fragments are separated by gel
electrophoresis, transferred to a membrane, and probed with a gene
probe.
One advantage of this fingerprinting technique is that all bands are bright
(from chromosomal DNA) because they are detected by a gene probe.
AP-PCR, ERIC-PCR, and REP-PCR all have bands of variable
brightness and also can have ghost bands.
For PCR products a simple fragment pattern can be distinguised
immediately on a gel. This is used to confirm the PCR product or to
distinguish between different isolates based on restriction cutting of the
16S-rDNA sequence “ribotyping”. Also developed into a diversity
measurement technique called “TRFLP”.
Recombinant DNA techniques
Cloning – the process of
introducing a foreign piece
of DNA into a replication
vector and multiplying the
DNA.
Recombinant DNA - foreign
DNA inserted into a vector.
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These approaches are used to:
1. Find new or closely related
genes
2. Insert genes into an
organism, e.g., an
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3. Produce large amounts of
a gene
Cloning
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Metagenomics
Genetic analysis of an entire microbial community.
Metagenomics involves the cloning of large fragments of DNA extracted from the
environment, allowing analysis of multiple genes encoded on a continuous piece
of DNA as well as allowing screening of large environmental fragments for
functional activities.
Two main approaches:
• sequence analysis of all DNA present
advantage: allows unparalleled access to the genetic information in a sample
disadvantage: difficulty in organization and interpretation of the sequenced
information obtained from complex communities
• directed sequencing for identity (16S rRNA gene or a functional gene)
advantage: allows rapid access to specific identity or functional data from an
environmental sample
disadvantage: provides more limited information about the sample
DGGE Analysis
DGGE – denaturing gradient gel electrophoresis
DGGE is a way to separate multiple PCR products of the same size. These
products can be generated by a 16S-rRNA PCR of community DNA.
DGGE uses either a thermal or a chemical denaturing gradient to separate
bands on the basis of their G+C content.
Once the bands are separated they can be sequenced to allow
identification. The banding patterns themselves can be used to evaluate
whether changes in the population are taking place.
Note of caution: PCR is inherently biased, some primers work better with
some target sequences than others and primers will preferentially amplify
targets that are present in high concentration. So scientists still don’t know
how accurately this type of analysis depicts the population actually present.
TRFLP Analysis
TRFLP = (terminal restriction fragment length polymorphism analysis)
•
A way to separate multiple PCR products of the same size. These products
can be generated by a 16S-rRNA PCR of community DNA
•
The PCR is performed as usual with two primers, but one is fluorescently
labeled
•
The PCR products are then cut up using a restriction enzyme
•
The fluorescently labeled PCR pieces are detected
•
TRFLP steps:
1. Extract community DNA
2. Perform 16S rRNA PCR using fluorescently-labeled primer
3. Choose a restriction enzyme for TRFLP that will give the greatest diversity
in restriction product size
Gel
electrophoresis
analysis
Automated DNA analyzer
0.10
Relative Abundance
0.08
0.06
0.04
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100
200
300
400
Fragment Length
500
600
700
Some approaches for analysis of the various bacterial communities
present in environmental samples
1. Culture and identify via 16S-rRNA PCR and sequencing
2. Extract DNA, subject to 16S-rRNA PCR, clone, then sequence
“clone libraries”
3. Extract DNA, subject to metagenomic analysis
4. Extract DNA, subject to 16S-rRNA PCR, then DGGE analysis
5. Extract DNA, subject to 16S-rRNA PCR, then TRFLP analysis
Discuss the advantages and disadvantages of each of these approaches
Reporter genes
Reporter genes are genetic markers that are inserted into the organism of
interest to allow easy detection of the organism or its activity.
insert
reporter
gene
Examples of reporter genes: lux genes (luminescence), gfp genes (green
fluorescent protein), beta-galactosidase gene (produces blue color).
Microarrays
Constructed using probes for a known nucleic acid sequence or for a series of
targets, a nucleic acid sequence whose abundance is being detected.
GeneChip microarrays consist of small DNA fragments (referred to also as
probes), chemically synthesized at specific locations on a coated quartz
surface. By extracting, amplifying, and labeling nucleic acids from experimental
samples, and then hybridizing those prepared samples to the array, the amount
of label can be monitored at each feature, enabling either the precise
identification of hundreds of thousands
of target sequence (DNA Analysis) or the
simultaneous relative quantitation of the
tens of thousands of different RNA
transcripts, representing gene activity
(Expression Analysis).
The intensity and color of each
spot provide information on the
specific gene from the tested
sample.
Affymetrix gene arrays for specific organisms:
Arabidopsis Genome Arrays
B. subtilis Genome Array (Antisense)
Barley Genome Array
C. elegans Genome Array
Canine Genome Array
Drosophila Genome Arrays
E. coli Genome Arrays
Human Genome Arrays
Mouse Genome Arrays
P. aeruginosa Genome Array
Plasmodium/Anopheles Genome Array (malaria)
Rat Genome Arrays
S. aureus Genome Array
Soybean Genome Array
Vitis vinifera (Grape) Array
Xenopus laevis Genome Array
Yeast Genome Arrays
Zebrafish Genome Array
Microarray technology is developing fast beyond pure culture:
In 2005, arrays are containing > 250,000 probes.
In 2006, arrays are containings > 500,000 probes.
Microarray analysis is developing the next generation of chips to examine
“who” is in environmental samples and “what” they do:
Phylochip is a microarray with DNA signatures for 9000 known species in the
phyla of Bacteria and Archaea to examine “who” is there.
Geochip is a microarray with DNA signatures for various functional genes to
examine “what” functions are present