Transcript PCR - Polymerase Chain Reaction

Culturing techniques
• Microbiology owes it’s roots and much
information we know about some organisms
from culturing techniques
• Culturing generally attempts to develop an
isolate (not always of course)
• Choose medium, environment (chemical and
physical) – innoculate and grow…
• Plating or streak culturing – smear a sample
so it becomes diluted…
Most Probable Number (MPN)
• Culturing technique that provides both cell
density information and function (grouped
into cells that can metabolize a certain way)
http://www.i2workout.com/mcuriale/mpn/index.html
Environmental Sampling
• As soon as you remove organisms from their
surroundings, they DO NOT just stop acting – often
changing function and ecology quickly
• ‘Fixing’ is the technique of preserving the cells as
close to their actual distribution as possible
– Physical (freezing)
– Chemical (additives to arrest function)
– These techniques kill all, or at least most, cells
• Preservation – most also preserve the material you
are after until you get to the lab
Materials
• Fixation:
– Freezing – How fast? Dry ice-ethanol, liquid nitrogen
– Chemical – Ethanol, Glutaraldehyde,
Parafomaldehyde
• Preservation
–
–
–
–
Freezing
Ethanol
RNAlater
Salts, buffers
Sterile technique
• When should we be MOST concerned with
maintaining sterile technique?
– SEM samples
– PCR samples
– FISH samples
– MPN samples
– Culture samples
– Shotgun samples
Primer Selection
• For any process, the primer selected for
use in PCR to amplify some piece of DNA
is the first point that determines what sort
of information you will be working with
• Universal for ID, targeted for group
selection, gene group targeting (NOT
necessarily expressed though)
Bias
• Bias occurs for any reaction – some
organisms will be selected over others in a
way that changes their apparent
abundance
• PCR – selects for organisms in higher
abundance (not good to ID organisms at
0.x-5%, roughly) some organisms
hybridize to primers faster, conditions
through PCR reaction can affect…
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’-TTC3”-AAG5’-TAATTT
3’-ATTAAA
Enzyme X
5’-TTC3”-AAG-
5’-CCGTTAGTT
3’-GGCAATCAA
5’-CAAGCGTTAGGACC
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
• Advantages
– Relatively easy to do
– Results can be banked for
future comparisons
• Limitations
DGGE
• Advantages
– Very sensitive to variations in
DNA sequence
– Can excise and sequence
DNA in bands
• Limitations
– Less sensitive phylogenetic
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
RFLP/DGGE info
• Grouped into sequences with SIMILARITY,
not necessarily identical!
• Operational Taxonomic Units (OTU) is the
simplest grouping
• Can compare these sequences to others
prepared in same way (same primers for
PCR, same restriction enzyme(s) )
– http://www.oardc.ohio-state.edu/trflpfragsort/
– http://mica.ibest.uidaho.edu/digest.php
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
sitehybridisation
hybridization
Fluorescent
(FISH) using DNA probes
Probe
(- 20 bases)
TA GC TG G C A G T
C G UAUCGAC C G UC A
UA
*
DNA
16S rRNA
Fluorescein
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
FISH-MAR
• Combine FISH to tag specific
organisms with
autoradiography
• FISH ID’s the organism based
on oligo hybridization
• Isotopically labelled substrates
are incorporated into the
organism showing metabolism
Single cell DNA extraction
• Using laser calipers, possible to isolate
and move a single cell, lyse, and extract
DNA from just that cell for amplification…