Fundamentals: Nucleic acids, DNA
replication, transcription, translation and
application to molecular detection
Prokaryotic cell
Binary Fission
• Bacteria reproduce asexually via
binary fission
• Each daughter cell is an identical
copy (or clone) of its parent cell
Microbial evolution 101
Generation 1
Generation 2
Generation 3
Generation N
Ancestor Genotype
Clones
Clones
Clones and
Divergent
Genotypes
Microbial genetics 101
• What is DNA ?
• What types of DNA molecules
are present in a bacterial cell?
• What’s the size of the genetic
material for a typical bacterial
pathogen ?
• How many genes does a
bacterial pathogen have ?
• What’s the average size of a
bacterial gene ?
What Is DNA?
Double Helix Structure and Antiparallel Orientation
Nucleotide = 5 carbon sugar +
nitrogenous base + phosphate group
DNA is a polynucleotide
Constituents of a Gene
•
•
•
•
5’
Promoter
Ribosome binding site
Open reading frame
Start & Stop codons
-35
Start
codon
Stop
codon
ATG
TAG
-10
Promoter Ribosome
binding
site
3’
The Central Dogma
DNA
DNA replication
Molecular
methods
Transcription
mRNA
Translation
Classical
methods
Protein/Enzymes
Post-translation
Toxins & other metabolites
DNA Replication
• Topoisomerases remove superhelicity
• New DNA is synthesized in the 5’ to 3’
direction
• Replication begins at a the origin of
replication (ori)
• Two replication forks proceed around
the chromosome (bi-directional) until
they encounter termination (ter) sites
• Replication is continuous on one
strand and discontinuous on the other
strand
• Chromosomes partitioned into two
daughter cells during cell division
DNA Replication
Leading strand;
continuous
replication
Lagging strand;
discontinuous
replication
• Helicase; unwinds helix
• Single-stranded binding protein; binds single-stranded DNA prevent
hybridization
• Primase; lays down RNA primers needed for DNA polymerase activity
• DNA polymerase I; remove RNA primers replace with DNA
• DNA polymerase II; DNA repair
• DNA polymerase III; major replication enzyme forms phosphodiester bonds
• Ligase; seals nicks by linking free 3’ OH with 5’ adjacent phosphate group
• Proofreading (DNA pol I & III) 3’ to 5’ exonuclease activity to remove incorrect
base
• Incorrect base incorporated every 1x108 to 1x1011 bases
DNA Polymerase and PCR
• DNA polymerase III
• Polymerase with or without 3’ to 5’ exonuclease
proofreading activity
– Taq (5’ - 3’ exonuclease activity); only degrades double
stranded DNA while extending
– Vent (3’ to 5’ exonuclease activity; specialized Taq)
– Implications
• Detection/subtyping methods
• Cloning
DNA Replication and Application to Molecular
Detection
• Polymerase chain reaction (PCR) or “DNA
photocopying”
– Simulate the natural DNA replication process to make
copies of DNA in vitro
– Make many copies of specific DNA fragment(s) in vitro
• Template, deoxynucleotidetriphosphates, primers, DNA
polymerase, enzyme cofactors, and buffer
RNA v. DNA
RNA in the cell
• Ribosomal RNA (rRNA) bulk of RNA in a cell
– 3 types (16s, 23s, and 5s)
– 3,000 copies in a cell
– Ribosomes; protein assembly during translation
• Messenger RNA (mRNA) 5-10% of RNA in a cell
– Almost as many types as there are genes
– Not stable in the cell; highly transcribed genes have a few hundred
copies; half-life a few minutes (1 to 7 min.)
– Synthesized from DNA during transcription
– Move information contained in DNA to translation machinery
• Transfer RNA (tRNA)
– About 50 types
– Pick up amino acid & transport to ribosome during translation
• Small RNA (sRNAs)
– 50 - 200 nucleotides
– Regulatory roles (e.g., affect mRNA stability and translation)
The Central Dogma
DNA
DNA replication
Molecular
methods
Transcription
mRNA
Translation
Classical
methods
Protein/Enzymes
Post-translation
Toxins & other metabolites
Transcription and translation are coupled
in bacteria
Holoenzyme (RNA
polymerase and
sigma factor
5’
3’
tRNA
Anticodon
Transcription
mRNA
Ribosomes
Translation
Direction
Translated
Protein
3’
5’
50S large
subunit (23S and
5S RNA and
proteins)
30S small
subunit (16S
RNA and
proteins)
A closer look at transcription
• DNA used as template to synthesize
complementary mRNA molecules
• RNA polymerase (pol) binds to promoter
region in double-stranded DNA
• Sigma factors help RNA pol bind promoter
& target genes to be transcribed
• -10 and -35 region 5’ of transcription
start site
• Local unwinding of double-stranded DNA
• RNA pol recognizes transcription start site
• RNA pol adds nucleotides 5’ to 3’
• RNA pol termination RNA pol and RNA
molecule released
•Rho-dependent
•Rho-independent (hairpin loop;
termination sequence)
The Central Dogma
DNA
DNA replication
Molecular
methods
Transcription
mRNA
Translation
Classical
methods
Protein/Enzymes
Post-translation
Toxins & other metabolites
A closer look at translation
• Three ribosome sites:
• A site; entry of aminoacyl tRNA
(except 1st aminoacyl tRNA or start
codon, which enters at P site)
• P site; peptidyl tRNA is formed
• E site; exit for uncharged tRNA
• Shine-Dalgarno sequence or ribosome
binding site recognized (5-10 bases
upstream of start codon)
• Assembly of small and large ribosome
subunits
• Amino acids added to carboxyl end of
growing chain
• Protein exits ribosome through tunnel
in large subunit
• Termination occurs when one of three
termination codons moves into A site
Genetic code
Application to molecular detection
in food microbiology
• Molecular detection methods include assays that target
nucleic acids (i.e., DNA and RNA)
• DNA detection methods
– Detect presence or absence of gene(s) or gene fragment(s)
specific to the target organism
– Detection of universal gene or gene fragment (e.g., 16s rRNA)
followed by DNA sequencing
– Detection of DNA does not differentiate between viable and nonviable organism
• mRNA detection methods
– mRNA is rapidly degraded and detection indicates presence of
viable organism
PCR Applications
• PCR detection particularly useful when
– Classical detection too time-consuming
– Differentiation from closely related non-pathogenic
organisms is difficult
• Listeria monocytogenes
– Only species in Listeria genera that is pathogenic to
humans
– PCR assay targeted to detect hemolysin (hlyA) gene can
detect presence and differentiate L. monocytogenes
from other Listeria spp.
PCR Reaction Components
•
•
•
•
•
•
•
1 - Small quantity of DNA added to tube
2 - DNA polymerase
3 - Oligonucleotides (primers)
4 - Deoxynucleotidetriphosphate bases
5 - MgCl2
6 - Buffer
7 - Sterile ultrapure water
Polymerase Chain Reaction (PCR)
Fundamentals
DNA Extraction
Introduction to PCR
• DNA  Genetic information for every animal, plant and
microorganism
• Unique variations in DNA allow us to track it back to the
organism it originated from with precision
• Comparative genomics, forensics, fingerprinting  often
require significant amounts of DNA
• PCR can synthesize, characterize and analyze any specific
piece of DNA
What is PCR ?
Polymerase Chain Reaction:
– in vitro (DNA synthesis in a tube)
– Yields million of copies of target DNA sequence
– Repeated cycling action
– Involving DNA polymerase enzyme
PCR Principles
• Conceptualized by Kary Mullis in 1983
• DNA amplification in vitro using the following
components:
– Two synthetic oligonucleotides (primers)
complementary to each end of targeted DNA sequence
– Single nucleotide bases as substrate
– DNA polymerase; a naturally occurring enzyme
responsible for in vivo DNA replication and repair
PCR Applications
• Food Science:
– Detection or molecular confirmation of specific
microorganisms present in foods
– Molecular subtyping of isolates
• Molecular Biology:
– Mutagenesis, cloning or sequencing
• Evolutionary Biology:
– Re-create the evolutionary history of a group of taxa
PCR Applications
• PCR detection particularly useful when
– Classical detection too time-consuming
– Differentiation from closely related non-pathogenic
organisms is difficult
• Listeria monocytogenes
– Only species in Listeria genera that is pathogenic to
humans
– PCR assay targeted to detect hemolysin (hlyA) gene can
detect presence and differentiate L. monocytogenes
from other Listeria spp.
PCR Reaction
• High temperature “melts” double strand DNA
helix into single strand DNA
• Two synthetic sequences of single stranded DNA
(18-24 bases) known as primers target a region of
genome
• Forward primer and Reverse primer flank the
region of interest (usually <1000 base pair)
PCR Reaction Components
1 – DNA template
2 – DNA polymerase
3 – Primers (oligonucleotides)
4 – Deoxynucleotidetriphosphate bases (dNTPs)
5 – MgCl2
6 – Buffer
7 – Sterile ultrapure water
PCR Reaction Set-up
1 - DNA Template
– Theoretically, PCR can detect as little as one DNA
molecule
– Template DNA should be present in small amounts (<
106 target molecules; 1 ng of E. coli DNA = 3x105)
– Bacterial cells need to be lysed to make their DNA
accessible for PCR
PCR Reaction Set-up
2 – Primers
– Should be in great excess to template DNA to
ensure that most strands anneal to a primer
and not each other
– Generally between 0.1 and 0.5 µM optimal
concentration
– Higher primer concentrations may cause nonspecific products
PCR Reaction Set-up
3 - Deoxynucleotide Triphosphates (dNTPs)
– Building blocks of DNA A’s, T’s, G’s, & C’s
– Essential to have enough dNTPs to make
desired number of target sequence copies
– Final concentration of EACH dATP, dTTP, dCTP
and dGTP should be 0.1 mM
PCR Reaction Set-up
4 - DNA Polymerase Enzyme:
– Original PCR performed with E. coli DNA
polymerase, but high temperature (94-95oC)
needed to denature double stranded DNA also
denatured this polymerase
– Problem solved using thermostable Taq DNA
polymerase
– Concentration should be around 0.5 to 5
units/100µl reaction volume
PCR Reaction Set-up
5 - MgCl2
– Essential for optimum Taq activity and for
proper primer annealing
– Excessive concentrations cause non-specific
products
– Standard PCR reaction contains between 1.5
and 5.0 mM MgCl2
PCR Reaction Set-up
6 - PCR buffer
– Buffer keeps the PCR reaction at the proper pH
(6.8-7.8) for enzyme activity
– Contains 10-50mM Tris-HCl and KCl or NaCl (50
mM)
– Salt also helps to stabilize hybridization of
primer to target DNA
PCR Reaction Set-up
7 – High quality water
– Dilutes template, buffers etc.
PCR Demo Cycling Action
• http://www.dnalc.org/files/swfs/animationlib
/pcr.exe
Standard Thermal Cycling Conditions
25-40 cycles
*Denaturation
94-96°C 94-96°C
2-10 min 1-2 min
*Initial
denaturation
*Annealing
50-55°C
1-2 min
*Extension
72°C
1-2 min
72°C
5-7 min
*Final
extension
4°C
∞
• Newly synthesized extension product of one primer serves as
template for annealing of the second primer in subsequent
cycles
• Each reaction cycle doubles the number of DNA copies or PCR
products
Amplification Cycles
• Initial denaturation at 94-96oC for 2 min
• Standard PCR protocol has 20-45 cycles of the following
time/temperature combinations
• 1-2 min @ 94-96oC
– Denature DNA
• 1-2 min @ 50-55oC
– Primers hybridize by forming hydrogen bonds to complementary
sequence
• 1-2 min @ 72oC
– DNA polymerase binds and extends a complementary strand from
each hybridized primer
• One final hold at 72oC for 5-7 min optional for higher
product yield
Exponential Amplification
• Newly synthesized extension product of
one primer serves as template for
annealing of the second primer in
subsequent cycles
• Each reaction cycle doubles the number of
DNA copies or PCR products
Important PCR Innovations
• Acquisition of heat stable DNA polymerase
(Taq) from Thermus acquaticus which
inhabits hot springs
– Remains active during repeated denaturation
cycles
• Thermal cycler or thermocycler
– Computer controlled heating block for
repetitive temperature change cycles required
for PCR reaction
Basics of DNA Extraction
Goal  Gain access to DNA
1) Classic DNA Extractions methods
2) Silica membrane (commercial kit)
3) Microwave (yep, just nuke the sample)
Classic DNA Prep
1. Disrupt the cell wall
– Mechanically Beadbeating, sonication, French
press
– Enzymatically  Lysozyme
2. Remove membrane lipid
– Triton-X, Sodium dodecyl sulfate (SDS)Detergents
3. Degrade proteins
– Proteinase K
4. Extract residual proteins  phenol:chloroform
extraction
5. Ethanol precipitation
Commercial Silica Column DNA
Purification
1.Enzymatic lysis &
protein degradation
2.Apply supernatant to
silica membrane in a
column
3.DNA is negatively
charged; silica
membrane is
negatively charged
Na+
Commercial Silica Column DNA
Purification , cont.
4.High salt and Ethanol washes rinse
degraded protein through the column
5. Elution Water with low salt
concentrations disrupt the
DNA-Na-Silica interaction releasing the
DNA from the column
Microwave
• Colony PCR or “dirty lysates”
1. Select an isolated colony from an agar plate
2. Transfer a portion to a PCR tube (0.2 mL
tube)
3. Microwave Time dependent!
4. Add water
5. Add PCR reagents & thermal cycle
Concepts of Primer Design
• Design is crucial to successful amplification of
target DNA by PCR
• Determine the size and location of PCR product
• Well-designed primers can deter amplification of
background and non-specific products
• Poorly designed primers result in no or very low
PCR product yield
• Several computer programs are available for
selecting primers
Concepts of primer design
Goal Balance specificity and efficiency of
amplification
Primer selection/analysis software assess these two
criteria by evaluating the following criteria:
–
–
–
–
–
Primer length
Terminal nucleotide
G + C content and Tm
PCR product length
Placement in target
sequence
Concepts of primer design
• Primer Length:
– Primers between 18-24 nucleotides in length tend to
be very sequence-specific if the annealing temperature
is set within a few degrees of the primer Tm
– Optimize PCR by using the minimum primer length that
ensures Tm of 54oC or higher
Concepts of primer design
Terminal Nucleotides:
– 3’ terminal positions are essential for controlling mispriming
– 3’ end of primers must be carefully selected to prevent
homologies within the primer pair known as primerdimer where the PCR product is amplification of
primers
GC content and Tm
– Primers with 50% G+C content have a Tm between 5662oC
– Tm and GC content should be similar between primer
pairs
– A/T = 2°C
– G/C= 4°C
Concepts of primer design
• Use multiple sequences to design/validate
primers if possible
– WHY?
• PCR Product Length and Placement within Target
Sequence
– Length of PCR product affects efficiency of
amplification
– Size of the PCR product depends on application
Degenerate primers
• Primers with mixed base pairs
Isolate 1
Isolate 2
Isolate 3
Isolate 4
5’ ATGGCATCTGACTGACACCACCTCAATCAA 3’
5’ ATGCCATCTGACTGACACCACCTCAATCAA 3’
5’ ATGGCATCTGACTGACACCACCTCAATCAA 3’
5’ ATGGCATCTGACTGACACCACCTCAATCAA 3’
Primer sequence
5’ ATG(G/C)CATCTGACTGACACC 3’
50% of primer synthesized with each nucleotide
• Inosine
Isolate 1
Isolate 2
Isolate 3
Isolate 4
5’ ATGGCATCTGACTGACACCACCTCAATCAA 3’
5’ ATGGCATCAGACTGACACCACCTCAATCAA 3’
5’ ATGGCATCTGACTGACACCACCTCAATCAA 3’
5’ ATGGCATCCGACTGACACCACCTCAATCAA 3’
Primer sequence
5’ ATGGCATC(I)GACTGACACC 3’
Concepts of primer design
• Hypothetical DNA and primer sequences
5’ ATGCCGCAATTCGTTATTACTTCGATCCG 3’
*reverse primer 3’… TAGGC 5’
5’ ATGCC … 3’ *forward primer
3’ TACGGCGTTAAGCAATAATGAAGCTAGGC 5’
5’-3’ primer sequences
F - atgcc
R - cggat
Primer design exercise
• You will be provided a hand-out with the DNA
sequence for Salmonella enteritidis invA
• Design a set of primers to amplify a 600 base pair
region of this gene
• Write down the sequence of both your primers in
the 5’ to 3’ orientation (requested by primer
synthesis companies)
• Calculate G+C content for your primers to make
sure they are similar
Lecture 34 PCR Product Detection
FS 362
Components in a PCR Reaction
•
•
•
•
•
•
•
Cycling Temperatures
25-40 cycles
*Denaturation
?°C
?°C
2-10 min
1-2 min
*Initial
denaturation
*Extension
*Annealing
?°C
1-2 min
?°C
?°C
1-2 min
5-7 min
*Final extension
?°C
∞
DNA Quantity & Quality
• Presence/Absence of DNA target and PCR
product  Agarose Gel Electrophoresis
• Quantity of DNA target & PCR  Nanodrop,
Bioanalyzer
• Quality of PCR product  Bioanalzyer,
Nanodrop
PCR Product Evaluation
Before a PCR product is used in further applications
(e.g., DNA sequencing) we need to make sure:
– There are bands; not every PCR is initially successful
and optimization is usually required
– The bands are the correct size; it is possible for
primers to anneal to an untargeted location on the
genome
– There is only one band per reaction; if primers fit on
other parts of the genome multiple non-specific bands
may be present
Detection & Analysis of PCR Products
• Agarose gel
electrophoresis
• PCR products
visualized when
stained Ethidium
Bromide (EtBr)
• EtBr intercolates
with DNA bases &
fluoresces upon
exposure to UV light
Agarose gels
• Cast by melting agarose in buffer until solution is clear
– Pre-cast gels are commercially available
• Gel casting tray contains combs to create wells
• Upon cooling, agarose solidifes
– Density of agarose matrix determined by concentration of agar in
solution
• Negatively-charged DNA migrates through the gel matrix
when electric field is applied.
DNA Migration in Agarose Gels
• Molecular size of DNA
– Larger molecules migrate more slowly than
smaller molecules
• Larger molecules experience more friction
• Larger molecules wiggle through pores in
agar matrix slower
• Agarose concentration
– Lower concentrations allow better separation of
large fragments
– Higher concentrations allow better separation of
smaller fragments
DNA Migration in Agarose Gels
• Voltage
– At low voltage, rate of DNA fragment migration is
proportional to voltage applied
– As voltage increases, range of fragment separation
decreases
• Electrophoresis buffer
– Buffers stabilize pH and provide ions for conductivity
– Composition and ionic strength of electrophoresis buffer
used to make agarose gel affects mobility of DNA
DNA Migration in Agarose Gels
• Molecular size of DNA
– Larger molecules migrate more slowly than
smaller molecules
• Larger molecules experience more friction
• Larger molecules wiggle through pores in agar
matrix slower
DNA Migration in Agarose Gels
• Voltage applied
– At low voltage, rate of DNA fragments migration is
proportional to voltage applied
– Range of separation of fragments decreases as voltage
increases
• Electrophoresis buffer
– Buffers stabilize pH and provide ions for conductivity
– Composition and ionic strength of electrophoresis
buffer used to make agarose gel affects mobility of
DNA
Staining DNA in Agarose Gels
• Ethidium bromide
– Contain planar group that intercalates between
stacked bases of DNA
– Ethidium bromide dye bound to DNA shows
increased fluorescence than dye in free
solution
– DNA can be detected in the presence of free
ethidium bromide in gel
Agarose Gel Electrophoresis of PCR
Products
pGEM N301 N302 N303 N304 N305 N306 N307 N308 N309 +con
-con
pGEM
DNA & Spectrophotometry
• DNA absorbs light at 260nm
• Protein absorbs light at 280nm
• Salts, phenol, EtOH absorb light at 230nm
Greater the absorbance, the greater the
concentration
• 260/280 ratio indicator DNA purity
• 260/230 ratio indicator of residual salts
Nanodrop Spectrophotometry
Nanodrop Results
Quality
• Spectrophotometry can’t determine if DNA
target or PCR product is dsDNA (intact DNA)
• Alternative methods are required to
determine DNA integrity  Agilent
Bioanalyzer
• Gel on a chip
Agilent Bioanalyzer Overview
Agilent Bioanalyzer
Listeria monocytogenes total RNA
PCR-based foodborne pathogen
detection methods & applications in
the food industry
FS 362
Review
• What is polymerase chain reaction (PCR) and
what is the purpose of this reaction?
• What role does temperature play in PCR?
• What role do primers play in PCR?
• Characteristics of well-designed primers.
• 5 ways to optimize a PCR reaction.
Today
• PCR platforms
• Advantages/Disadvantages of PCR detection
systems
• Characteristics of high quality assays
• Good Laboratory Practices
The Central Dogma
DNA
DNA replication
Molecular
methods
Transcription
mRNA
Translation
Classical
methods
Protein/Enzymes
Post-translation
Toxins & other metabolites
DNA-based detection by PCR
DNA
• Detects chromosomal or extra-chromosomal DNA
(i.e. plasmids)
• Advantages:
–
–
–
• Disadvantages:
–
–
DNA-based detection by PCR
DNA
DNA replication
Transcription
mRNA
Examples used in industry
• hly assay for Listeria monocytogenes
– Listeria spp. v. L. monocytogenes
• invA assay for Salmonella
– Salmonella v. non-Salmonella
• Multiplex for STEC
– Detection of multiple genes required to identify
strain likely to cause severe disease
Multiplex PCR
• Used to target multiple genes in a single test
– Advantages
• More information from a single test
• Use less reagents/consumables
• Save on time
– Disadvantages
• Requires time for optimization
• Single PCR reaction that contains multiple, unique primer sets
– Each primer set must amplify fragments of varying sizes specific to different
DNA sequences
– Annealing temperatures for each primer set must be similar in order to work
correctly within a single test
Multiplex & STEC
stx1 = 534 bp
stx2 = 384 bp
eaeA = 255 bp
EHEC hlyA = 180 bp
• stx2 > stx1
• eaeA required
• hlyA required
RNA-based detection by PCR
• Reverse-transcriptase
PCR
DNA
mRNA
Reverse
transcriptase
Protein/Enzymes
Toxins and other metabolites
cDNA
RNA-based detection by PCR
DNA
• Advantages:
–
–
• Disadvantages:
–
–
mRNA
Reverse
transcriptase
cDNA
Applied Biosystems Salmonella &
L. monocytogenes Rapid Detection System
Step 2
Step 3 RT-PCR
Step 4: Analysis
•
Real-time
PCR
Works just as conventional PCR does except:
– Involves fluorescent dye(s)
– Data collection throughout the PCR process
• Allows detection of target in real time during DNA
amplification
PCR Chemistries Used to Detect
Foodborne Pathogens
•
•
•
•
SYBR Green
Hydrolysis probe-based (e.g., TaqMan)
Molecular Beacon
Scorpion
SYBR Green
• Binds to double-stranded DNA
– Binds to minor groove of dsDNA
– Prefers G and C base pairs
• Light is emitted upon excitation
– Optimal excitation/emission wavelengths of 497/520 nm
• Advantages
– Inexpensive, easy to use
– Minimal effort to design
– Works well with a single defined target
• Disadvantages
– Binds to any double-stranded DNA
• Primer dimers
• Non-specific products
– Dissociation curve or melt curve must follow the PCR protocol
Melt curve analysis
EXAMPLE:
• BAX system for detection of
L. monocytogenes or
Salmonella
Idaho Technoloogy Inc.
Probe-based PCR: Principle of FRET
• Fluorescence Resonance Energy Transfer
• Quencher dye and reporter dye
– The excited reporter dye transfers energy to a quencher
dye rather than fluorescing
– Quencher dye must be in close proximity to a reporter
dye in order to have a “quenching” effect
Reporter/donor
excitation
Reporter
Quencher
Close proximity
Separation
Hydrolysis probe-based PCR
• Also referred to as TaqMan assay
• Takes advantage of 5’→3’ exonuclease activity of Taq
Labeled probe hybridizes to
DNA target sequence

Intact probe, the reporter
does not fluoresce

Taq cleaves the fluorogenic
tag from the probe

RT-PCR
Multiplex Real-Time PCR
Cy5
Texas Red
FAM
TET
Probe-based real-time PCR
• Advantages
– Increased specificity
– Able to detect multiple targets
– Allows for quantification
– Faster than conventional PCR
• Disadvantages
– Expensive to synthesize
PCR Controls
• PCR Controls
– Negative – Use water as the “template”
– Positive – DNA that will give the expected result
• Internal or amplification control
– Monitor for PCR inhibition
• Reverse-transcriptase PCR
– RT-negative control – Sample with no reverse
transcriptase enzyme
– Indicator for level of contaminating genomic DNA
How to evaluate PCR detection methods
(i.e., LIFE SKILLS)
• What is the target gene?
– How/why was it chosen
– What data are available to support the choice
• Virulence data? Sequence data?
• What is the target molecule?
– DNA? mRNA? rRNA?
– How is expression of mRNA assured?
• What is the assay and assay format?
– PCR, other amplification method, detection
without amplification (e.g., ACCUPROBETM)
How to evaluate molecular detection
methods II
• How was the assay validated
– Pure cultures, spiked foods, or “real samples”
• What isolates or samples were used
• What was used as “gold standard”
- What are the most likely reasons for false positives
and false negatives
- What does a false negative most likely mean (e.g., does it
suggest an avirulent variant)
- How does the assay perform in the presence of
competitive microflora
- What controls are included in the assay
- Internal positive control?
Good Laboratory Practices for PCR
• Separate pre-PCR and post-PCR activities
– Perform in physically separate areas
– Minimize potential contamination with amplified
product
– PCR can be set up in sterile hood to minimize
contamination issues.
• Use aerosol resistant pipette tips
– Use new pipette tips when transferring samples
– Prevent aerosolization when ejecting the tip
• Use powder-free gloves
– Change gloves frequently
GLPs for PCR
• Spin tubes (e.g., sample tubes) briefly to
bring contents away from lid
– Avoid touching rim/inside lids of tube to
ensure no cross-contamination occurs.
• Work slowly – do not rush
• Adequately chill cooling blocks before use.
• Minimize freeze/thaw cycles for frozen
reagents.
DNA Sequencing
DNA sequencing
• Biochemical process to determine of the order
of nucleotide bases
– Chemical degradation
• (Maxam and Gilbert, 1977)
– Enzymatic synthesis
• Sanger Method (Sanger et al., 1977)
• Generate sequences that begin at fixed point
and terminate at a particular type of residue
(A, T, G or C for Sanger Method)
DNA sequencing
Sanger method has served as the workhorse of
DNA sequencing projects for the past 30 years
– Human genome sequencing project
– >900 microbial genome sequences available
– Multiple genome sequences available for many
foodborne pathogens
• >20 genome sequences available for Listeria
monocytogenes
DNA sequencing
• Sequencing relies on DNA chain termination
by dideoxynucleotide triphosphates (ddNTPs)
Cycle sequencing demonstration
DNA sequencing
• Termination points are nucleotide specific but
occur randomly along length of target DNA
• Generates many fragments of different lengths
• Fragments resolved by electrophoresis
– Discriminate DNA’s that differ in length by as little
as a single nucleotide
– Four populations loaded into adjacent lanes of
sequencing gel, order of nucleotides along DNA
can be read from gel image
Classic Sanger sequencing
• Sequencing
performed in four
separate reactions
each containing one
of the four ddNTPs
• Run on adjacent lanes
in a denaturing
polyacrylamide gel
• Short runs and can be
difficult to interpret
Automation of Sanger Method
• Cycle sequencing/Dye-terminator
• 15-40 rounds of conventional thermal cycling
–
–
–
–
Denaturation of double stranded DNA
Annealing of primer to sequence
Extension of primer by thermal stable DNA polymerase
Termination of extended strand by ddNTP incorporation
• Fluorescent dyes to ddNTPs that become incorporated to
3’ end of sequencing reaction products
– Same primer in a single tube
• Capillary electrophoresis
Electropherograms
DNA sequencing success
• Key to consistent successful cycle sequencing
is cleanliness of DNA template
– Impurities (e.g., agarose, salts, proteins) cause
premature termination and pausing of DNA
polymerase
– Degraded DNA cause false priming
• DNA template should be purified and the
quantity and quality of DNA template should
be assessed prior to submitting samples for
sequencing
Illumina Sequencing by Synthesis
See link to URL on blackboard
Ion Torrent (Personal Genome
Maching (PGM))
http://www.iontorrent.com/thesimplest-sequencing-chemistry/
Pulse Field Gel Electrophoresis
Introduction to PFGE
• DNA
– Genetic information of an organism
• Chromosomal DNA
– Sequence specificity for each species and even strain
– Circular DNA molecule within bacterial cell
– Carries all “normal” genes employed for growth and other
practical functions
2
Introduction to PFGE
• Chromosome size variety of base pairs and genes for
bacteria:
Bacteria
Chromosome size (base pairs)
Estimated number of genes
Escherichia coli
4,639,211
4,279
Campylobacter
jejuni
1,641,481
1,654
Bacillus subtillus
4,214,814
4,112
Salmonella
Typhimurium
4,857,432
4,450
Listeria
monocytogenes
2,866,709
2,873
3
Introduction to PFGE
• Chromosome size variety of base pairs and genes for
L. monocytogenes:
L. monocytogenes
strain
Chromosome size (base
pairs)
Estimated number of genes
10403S
2,866,709
2,873
EGD-e
2,944,528
2,867
FSL J1-194
2,986,227
3,692
FSL-J2-071
3,149,923
3,373
FSL R2-503
3,001,696
4,767
• How does one differentiate between strains in a
rapid manner?
4
What is PFGE?
• Pulse Field Gel Electrophoresis
– Molecular method to produce a genetic “fingerprint” or
profile of a bacterial isolate
– Creates and visualizes segments of DNA from a bacterial
sample to be compared with other samples
– Achieved through breaking of chromosomal DNA into
segments by *restriction enzymes*
– Advanced method of gel electrophoresis
5
PFGE Background
• First introduced in 1984 by Schwartz &Cantor in
Cell 37:67-75
– Described a way to differentiate yeast chromosomes
• Segment chromosomal DNA, utilize non-uniform electric current,
compare DNA band profile
– Looked for a way to visualize segments of DNA
• Old size limit (50 kilobase)
• PFGE capability (10 megabase)
6
PFGE Applications
• Food Safety
– Epidemiological studies
– Tracking of outbreak strains
• Food Quality
– Monitoring subtle changes in fermentation cultures
• Yogurt, beer, wine industries
7
PFGE Applications
• Cancer Research
– Observations on dsDNA structure alterations by suspect
carcinogens
– Changes in DNA density for specific molecular weight regions
indicate structure alterations
• Genomics
– Cloning fragments to be sequenced can be separated using
PFGE
– DNA fingerprinting and physical chromosome mapping
– Location of promoter sites due to DNase sensitivities with
chromatin
8
PFGE Applications
• Epidemiological studies and strain differentiation
– Agencies such as PulseNet utilize PFGE to identify similar
or different bacterial strains to:
• Differentiate food-related clinical cases based upon suspect
pathogen strain
• Allows for accurate tracking of outbreak allowing for source
identification
• Goal of improved food safety for the public
9
Segmentation of DNA
• Nuclease – enzymes that breakdown strands of
nucleic acids
• Two ways to cut or “restrict” DNA to segments
– Exonuclease
• Works from the outside inwards, essentially dismantling DNA
piece by piece
• Systematically removes one nucleotide at a time from the end of
dsDNA
• Two versions of exonucleases (3’ to 5’ and 5’ to 3’) used to “clean
up” polymerase products and other processes
– Endonuclease
10
Restriction Endonucleases (RE)
• Cutting or restriction of dsDNA from within the
molecule
– RE works at a specific recognition site
• Recognition site is a specific sequence of nucleotides that are
identified and cut leaving fragments
11
Restriction Endonucleases
• RE’s read through dsDNA from 5’ to 3’ ends on both
strands (palindromes) until digestion site is recognized
• Thousands of different RE’s identified, some repetitive
(same recognition site as another RE, called
isoschizomers)
• Activity effected by
– pH
– Ionic strength
– Temperature
• Altered activity by changing the above conditions
results in slight alterations in recognition sites
(referred to as star activity)
12
Restriction Endonucleases
• Employed in PFGE to “randomly” break the
chromosome into fragments to be visualized
following electrophoresis
• The same RE is used for all samples to be compared
(should be the same bacterial species)
– Comparison between Listeria monocytogenes isolates from
the same outbreak would use ApaI or AscI (example RE’s)
13
DNA Orientation and Subsequent Digestion
RE Digestion of DNA
Supercoiled Chromosomal DNA
14
DNA Visualization through Gel Electrophoresis
• Traditional electrophoresis
– One dimensional application of electrical field
– DNA sample size < 50 kb can accurately be visualized
– Cause of method restrictions:
• One direction voltage application and strength
• 1.0% and higher agarose is too complex/thick to allow large
molecules (> 50kb) to move at different rates meaning one band
represents multiple segments
DNA Fragments
Gel
Particle
Pore
15
DNA Visualization through Gel Electrophoresis
• Pulse Field GE
– Two dimensional application of pulses
– Variation in direction of electrical fields
• These electric fields are altered throughout the same
electrophoresis run
• Time between shifts or pulses allows for reorientation
– Avoids the limitations of molecular sieving by forcing the
molecules to reorient themselves upon shifts in directions
• Application in one direction, pause, reorientation, application in
another direction, repeat
• Causing zigzag transversals
16
Comparison of Electrophoresis Fields
Traditional
_
PFGE
+
17
Field Inversion Gel Electrophoresis (FIGE)
• Periodical inversion of polarity of electrodes
– Shift in pulse application at 180°
– Molecules spend part of the time moving backwards
-/+
+/-
18
Transverse Alternating Field Electrophoresis (TAFE)
• Employment of two different electrode groups
creating simple geometrical pulse angles
• The pulse angle increases as the molecule moves
downwards
A (-)
B (+)
B (-)
A (+)
19
Rotating Gel Electrophoresis (RGE)
• RGE is a new form of PFGE
• Instead of having multiple or altering charges of
electrodes, the gel itself is moved
_
+
20
Clamped Homogenous Electrical Field (CHEF)
• Most commonly used
form of PFGE
• Applies multiple pulses
from varying sources to
create additional
vectors
• Varying angles result in
increased accurate
separation and more
clear resolution
21
Visualization
• Similar to PCR product visualization, following
electrophoresis, PFGE gels are:
– Stained with ethidium bromide
– Visualized through exposure to UV light
22
Pulse Field Gel Electrophoresis Performance
Review of PFGE Theory
• PFGE
– Production of a genetic “fingerprint”
• Creation of large genomic DNA fragments following restriction
enzyme digestion
• Such fragments are visualized following gel electrophoresis
• Comparing between lanes on the resultant gel allows for:
– Differentiation between strains
– Designation of same strain samples
2
Steps for PFGE
1)
2)
3)
4)
5)
6)
7)
Culture Preparation and Standardization
Sample Treatment
Sample Suspension/”Plug” Preparation
Restriction Enzyme Digestion
Pulsed Field Gel Electrophoresis
Visualization
Comparison
3
Culture Preparation
• Physical removal of a pure culture from agar plate
• Resuspension of culture in TE Buffer
• Standardization of culture suspension
– Standard quantity of bacteria/mL
– Less bacteria is more
• Increased lysis efficiency, increased resolution and sharpness of
bands, similar band intensities
4
Sample Treatment
• Cell suspension is treated to liberate DNA for further
manipulation
– Lysozyme for cell wall digestion
• Hydrolyzes the 1,4-β-linkages between Nacetylmuramic acid and N-acetyl-D-glucosamine
residues in the peptidoglycan layer
– Proteinase K for degradation of proteins and
inactivation of enzymes (DNases, RNases, etc.)
• Cleaves peptide bonds next to carboxyl groups of
amino acids
Egg – source of Lysozyme
Engyodontium album – mold
source of Proteinase K
5
Plug Preparation
• A “plug” refers to the physical suspension of the
lysed bacterial sample in mixture of:
– 1.5% Agarose
– 1% Sodium Dodecyl Sulfate
• Detergent aiding in cell lysis steps previously described
• Aids effectiveness Proteinase K
• Addition of agarose/SDS mixture to cell
suspension
– Can either have Proteinase K in cell suspension or plug
preparation
• Placement of mixture into a mold to form a plug
6
What is a “plug”?
• Comparison of plug
quality:
2mm width
7
Restriction Enzyme Digestion
• Restriction endonucleases employed to cut/digest
DNA molecules at specific points (recognition sites)
• Plug treatments
– Plug samples are submerged in RE mixture:
• RE
• Buffer
• Water
– Buffer type and treatment temperatures are
specific for the RE used
• Thorough washing following RE treatment following
treatment
8
Restriction Enzyme Digestion
• Reaction Conditions for high specificity (accurate restriction site
location)
– Buffer
• Provides adequate levels of ionic strengths (Na+, K+, Mg2+)
• ~pH 8.0
– Temperature
• Efficacy of digestion dependent on temperature of reaction
• Related to ideal temperature for organism RE was isolated from
– Ex: RE from a thermophilic bacteria has an ideal temp above 50˚C while an RE from E. coli has an ideal
temp at 37˚C
– Residual glycerol (>5%) effects reaction
• RE shipped in glycerol
– Concentration of RE
• Ideal ~ 40U
– Concentration and purity of DNA sample
• Incorrect or partial digestions
– Addition of Bovine Serum Albumin (optional)
• Protein shown to aid in binding and specificity
9
Restriction Enzyme Digestion
RE Concentration
10U
20U
40U
Asc1
Asc1
Apa1
Apa1
10
Restriction Enzyme Choices
• Overall goal of PFGE:
– Comparison of sample fingerprints to determine strain
identities and differentiation
• Accepted practice to do 2 different PFGE runs with at
least 2 different RE’s
• RE pairs for specific pathogens have been
determined and commonly used
– Ex: L. monocytogenes – AscI and ApaI
• Common RE’s published by PulseNet
11
Gel Preparation
• Thought PCR gel preparation and loading was challenging?
– Ensure level surface for pouring the gel
– Placement of plug into agarose gel
– Placement of plug onto comb prior to gel pouring (alternative)
• Following placement of plug in gel, fill in the hole with molten
agarose to seal off chamber hole
– Prevents the plug from floating out
12
PFGE Performance
• Finally, time to run the actual electrophoresis
• Components:
a.
b.
d.
c.
a.
b.
c.
d.
Gel Chamber
Pump
Chiller
Chef Mapper Pulse Source
13
Component Purposes
• Gel Chamber:
– Application of pulses from electrodes
– Hold gel in centralized locale
• Pump:
– Continuously moves recycled buffer through the chamber
• Chiller:
– Maintains temperature of buffer and therefore of gel in
chamber
• Chef Mapper Pulse Source
– Applies programmed pulses, times, application profiles
14
Important Parameters
• Pulse Angles
– Smaller angle of pulse application results in better
separation of larger DNA molecules
• Ex: 106˚ vs. 180˚
• Smaller molecules crammed together at the end of the run
– Larger angle of pulse application results in better
separation of smaller DNA molecules but no separation of
larger molecules
– Ideal compromise angle = 120˚
• Run Time
– 19 to 24 hrs
15
Important Parameters
• Switch Times
– Interval between
alternation of electric
fields
45 sec
60 sec
90sec
• Consider enough time for
molecule to reorient itself
• Switch Time Ramping
– Altering the amount of
switch times over the
length of the
electrophoresis run
• Ex: Initial switch time at
30sec; Final switch time at
120sec
– Linear vs. Non-linear
Ramping
16
Linear vs. Non-linear
17
• Voltage
Important Parameters
– Strength of electrical field (pulses) in V/cm units
• Large value – faster run, fine for small molecules
• Small value – slower run, better resolution for large molecules
• Compromise at 6 V/cm
– V/cm – Voltage applied over the distance between the electrodes
applying pulses
• Ex: 200V/33cm = ~6V/cm
• Buffer Concentration
– TBE 0.5X common
• Increase ionic strength = slower migration
• Decrease ionic strength = faster migration, poorer quality
• Buffer Temperature
– ~25˚C = fastest run times, poorest resolution and band separation
– ~4˚C = slowest run times, best resolution and separation
– ~14˚C = compromise, most commonly used
18
Visualization and Comparison
• Ethidium Bromide
staining and UV
visualization
– Same as PCR
• Comparison of lanes
– Use of BioNumerics
software to select
lanes and produce
statistical compatibility
19
Standards
• PCR
– Employment of a ladder standard to quantify band size
• PFGE
– Employment of universal strain as a standard
• Quantification of band size
• Comparison with other PFGE profiles previously run
• Allows for universal nature of gels and normalization on specific
band patterns
• Standard strain – Salmonella serotype Braenderup H9812
– Digested with specific RE (Xba1)
– Range of 20 to 1200kb
20
PFGE Conclusions
• PFGE is a common technique used to create a
genetic fingerprint of a sample (bacteria, yeast, etc.)
– Plethora of variability in parameters and conditions
– Standard procedures for conditions stated by PulseNet
• Genetic fingerprints are compared to determine if
samples are the same or different
• Generated similarity used to ID and track outbreak
strains as well as other applications
– PulseNet and other investigative bodies
21
Pulse Field Gel Electrophoresis;
PulseNet
PulseNet
• PulseNet:
– Responsible agency for investigating foodborne-related
outbreaks
– Employs PFGE to type and store all outbreaks in recent history
for comparison and epidemiological work
– Drives early ID of outbreak sources
– Standardizes all procedures for molecular fingerprinting
– Coordinated by the Center for Disease Control (CDC)
– Collaborates with:
• State and local health departments along with other federal agencies
(FDA, USDA, FSIS, CDC)
– http://www.cdc.gov/pulsenet/whatis.htm
2
PulseNet- Take on PFGE
• Positives
– More discriminating
than ribotyping
– Outside of RE choice,
entire process is
universal for all bacteria
– Proven to be very
reproducible
• Negatives
– Time-consuming
– High level of skill and
experience required
– Bands are not sequences
– Same bands are not
necessarily same DNA
sequence
– Relatedness are guides
not necessarily
phylogenic proof
3
Optimization
• PulseNet driving
improvement of PFGE
– Standardization
•
•
•
•
Protocols
Software
Equipment
Nomenclature of patterns
– Promote workshops for
PFGE use
– Annual update symposia
– Participants required to
be certified and trained
• Improvement to
PulseNet itself
– Increase participants
– Real-time subtyping and
communication
• Clinic to lab
• Lab to isolate
differentiation
• Isolate to profile
– Food industry?
– International?
4
PulseNet International
• International organization spawning from PulseNet in
the United States
• Agencies related to Public and National Health
Agencies in foreign countries
• Collaboration between nations and research groups
to investigate global outbreaks
5
6
PFGE Gel Interpretation
7
PFGE Gel Interpretation
8