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Genotypic Microbiological Methods
Can be used to determine genetic composition
of organisms:
Identify organisms (diagnostics)
Identify distinct groups of organisms
(taxonomy/systematics) by examining similarity
between organisms
Examine temporal or spatial variation of
populations
Study genes function and regulation
Assess viability of cells
Overview of Molecular (DNA) Methods
• Restriction Enzyme Digestion
– RFLP
DNA “fingerprinting”
– PFGE
• Labeled Probes
• PCR based methods
• DNA sequencing
• DNA microarrays
Combination of
methods e.g.
ribotyping
DNA isolation
•
•
•
•
Grow cells to stationary phase (for maximum yield)
Lyse cells with heat, enzyme, or detergent
Digest proteins with proteinase K enzyme
Separate DNA from other molecules by solubility, charge,
size etc..( methods vary)
Commercially available methods
Use centrifuge to pass cell lysate through filter, DNA
binds to filter
Add solvent to filter and centrifuge DNA out into
clean tube
Gel Electrophoresis
DNA samples added to wells in matrix
-
Gel made of translucent, porous matrix
+
DNA migrates at a rate inversely
related to log10 of amplicon size
Gel electrophoresis
EtBr binds to DNA as it
travels through the gel
EtBr fluoresces under UV
light
When viewed in the dark
UV light, the DNA appears
as bright bands
Larger fragments of DNA
remain near the top and
smaller ones migrate to the
bottom
Hybridization and Annealing of Nucleic
Acids
Complimentary sequences of ssDNA will bind together
to form dsDNA
Temperature at which dsDNA remains together
depends on percent of matching and GC content
Does not yield the DNA sequence of organisms, just
the sequence similarity between organisms
Total genomic hybridization can be used to estimate
overall genetic similarity between organisms
Oligonucleotide primers and probes can be designed to
detect and ID genes
Labeled Probes
Oligonucleotide-short piece of DNA
Complementary-has corresponding nucleotide base
sequence (ATCG- TAGC)
Target- specific region of organism’s DNA to be probed
Labels- a molecule that is attached to the probe and can
be observed by some direct (fluorescent) or indirect
means (immunodetection)
TATGCATT
TAACGCCGAGGTATGCATTGCATGCCTGACTGAATCGA
….ATTGCGGCTCCATACGTAACGTACGGACTGACTTAGCT……..
RFLP
RFLP-restriction fragment length polymorphisms
Organism B
Organism A
DNA
RE cuts wherever it
recognizes specific site
_
+
Gel electrophoresis
Separation and visualization DNA fragments
_
Large pieces of
DNA
Small pieces of
DNA
+
L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 L
Ribotyping
cells
DNA
EcoRI
Transfer to nylon
membrane
(Southern Blot)
Bind labeled
16S rDNA
Probe
Anti-probe Ab and
enzyme-linked color
reaction
1%
Agarose
Gel
+
Gel
electrophoresis
PFGE
(Pulse Field Gel Electrophoresis)
+
+
Agarose
gel
-
-
Repeatability and Discrimination
Trial 1
Trial 2
PCR
polymerase chain reaction
• Invented by Kary Mullis in 1983
• Now widely used for many types of scientific research
and medical diagnostics
• Works by amplifying target region of DNA using:
– synthetic oligonucleotides called primers
– Taq polymerase enzyme
– Temperature cycling
Concept
• Amplify small quantities of DNA by in vitro DNA
replication
Target DNA
PCR
Copies of Target DNA
(amplicons)
Generalized PCR cycle
repeated ca. 40 times
94 degrees
Celsiusdenaturation
72 degrees Celsiusextension
Ca.45-60 degreesprimer annealing
Taq
Target
sequence
Primers
3’…GTATTATGGTATGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA…
5’TACGAACGG
primers
3’CCATCGAAA
5’…TATCGAACGGAGACTTACTCTTATACCGTGGTAGCTTTGTAATGATATT…
Specificity of PCR depends on
the sequence to which they
bind
Extension (polymerization)
3’…GTATTATGGTATGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA…
5’TACGAACGG
Taq
Taq
3’CCATCGAAA
5’…TATCGAACGGAGACTTACTCTTATACCGTGGTAGCTTTGTAATGATATT…
PCR
Primers anneal to both strands of target sequence
New strands of DNA are added (5’ to 3’) from the primers
In subsequent cycles, primers can bind to amplicons in addition to the
original DNA
Amplicons increase exponentially
with each cycle
Single copy of
dsDNA target
1st Cycle
2nd
Cycle
3rd Cycle
Factors that influence specificity
• Stringency of conditions
– Degree of primer sequence match to target sequence
– Primer length
– Annealing temperature
• Uniqueness of target sequence
– Primers will bind wherever there is a complementary
target sequence
Factors that influence sensitivity
• Presence of necessary components
– Taq
– dNTPs
– Magnesium
– Target sequence
– Primers
• Presence of inhibitory chemicals
• Primer hairpins and self dimers
Can be used to generate qualitative or
quantitative data
L
1
2
3
-
Positive
Charge
Negative
Charge
L
1
2
3
-
fluorescence
Real Time PCR
Time
Can be used to estimate the
starting concentrations of DNA
Reverse Transcriptase PCR
• RNA converted to cDNA by reverse transcriptase
enzyme
• cDNA used to perform PCR
• Used to detect specific RNA viruses
• Used to test viability of cells
DNA sequencing
• DNA usually in the form of PCR amplicon
• One strand at a time
• Most thorough method of studying variation in
DNA and assessing DNA similarity
• Relatively expensive and time consuming
(however, this is constantly improving)
• Can now sequence entire genomes of organisms
Extension (polymerization)
3’TAGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA…
5’ATCGAACGGAGACTTACTCTTA T
Taq
dNTPs are randomlyincorporated into new
strand until a ‘stop’ is
added
T
A
A
G
A
C A
G
T
C
T
A
3’TAGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA…
5’ATCGAACGGAGACTTA
Taq
Possible fragments
A
G
C
T
T
A
G
T
If there is contradictory info, it
will be read as ‘N’
Sequence Trace
DNA microarrays (Gene Chips)
Labeled complementary nucleic acid binds and
stimulates chip
~1cm
Microarray Chip containing thousands of blocks
Each block is
affixed with
numerous copies
of a nucleic acid
sequence