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Molecular Biology 101 forLaboratory Professionals:
Part Two
Erik MunsonClinical Microbiology
Wheaton Franciscan LaboratoryWauwatosa, Wisconsin
The presenter states no conflict of interest and has no financial relationshipto disclose relevant to the content of this presentation.
1
OUTLINE
I. Cell biology vignette
II. Molecular diagnostic application
III. Life-creating, life-changing events
A. DNA structure
B. DNA replication
C. DNA replication
D. Transcription2
ReviewReview
3
COMPONENTS
DNA
Phosphate
O
O-
PO-
O-
O
HOCH2
2’Pentose(deoxyribose)
Base(thymine)
OH H
O
N
O
CH3
NH
O
2’
4
COMPOSITE
O H
O
CH2
O
O-
PO O-
-
NN
N
N
N H
H
Adenine
OH H
O
CH2
O
PO O
N
O
N
N
H
H H
Cytosine
5
N
N
N
N
N H
H
N
CH3
NH
O
Adenine ThymineH
HYDROGEN BONDING
N
N
N
N
O
N HH
H
O
N
O
N
NH
H
Guanine Cytosine
H
Hketo group amino group
6
2
UNWIND DOUBLE HELIX
7
POLYMERASE SPECIFICITY
Catalyze ester bond ONLY between first5’ phosphate of new nucleotide and3’ hydroxyl of previous nucleotide
Polymerase ONLY allows addition ofphosphate to pre-existing hydroxyl
5’ to 3’ direction
8
OH H
O
CH2
O
O-
PO O-
NN
N
N
N H
H
Adenine
O-
5’
’
3’
( )
Cytosine
O
PO O-
N
O
N
N
H
H H
OH H
O
CH2
5’
3’
DNApolymerase( ) x 3
9
Molecular Diagnostics ClassificationsMolecular Diagnostics Classifications
10
Probe anneals to target of interest
NUCLEIC ACID HYBRIDIZATION
TARGET DETECTProbe
11
PROBE TECHNOLOGY
More effective on colonial growth than onprimary clinical specimens
12
3
Amplify target of interest prior to detection
NUCLEIC ACID AMPLIFICATIONTESTING (NAAT)
TARGET
Primer
PrimerDETECTAMPLIFY
13
ANALYTICAL SENSITIVITY
Manual of Clinical Microbiology, 6th edition; 1995 14
Denature(95°C, 5 min)
Anneal/hybridize(30°C 2 min)
PROTOCOL (Mullis et al.)
(30 C, 2 min)
Repeat 19 times;add Klenow each time
Klenow extension(30°C, 2 min)
15
Taq polymerase isolated from extremethermophile Thermus aquaticus
Thermostability eliminates necessity toreplenish enzyme with each new cycle
REVOLUTIONARY FINDING
Science 239: 487-491; 1988 16
Denature (95°C)
Anneal/hybridize (62°C)
“OPTIMIZED” PCR PROTOCOL
~40 cycles
Extension (72°C)
17
Elapsed time: 3 hours
18
4
Elapsed time: 4 hours
19
Elapsed time: 4.5 hours
20
Elapsed time: 6.5 hours
21
Elapsed time: 7 hours
22
Elapsed time: 5.5 hours
Alternatively…23
OLD
SCHOOL 24
5
25
Diagnostic Application: Real time PCR
Elapsed time: ~1 hour
Diagnostic Application: Real‐time PCR
26
Surface area to volume ratio
HOW DOES THIS HAPPEN?!?!
27
Surface area to volume ratio (increased)
HOW DOES THIS HAPPEN?!?!
28
Real-time detection
Surface area to volume ratio
HOW DOES THIS HAPPEN?!?!
No target control
29
Real-time detection
Surface area to volume ratio
HOW DOES THIS HAPPEN?!?!
BASELINE
30
6
Real-time detection
Surface area to volume ratio
HOW DOES THIS HAPPEN?!?!
BASELINE
31
Cycle threshold (CT)Real-time detection
Surface area to volume ratio
HOW DOES THIS HAPPEN?!?!
BASELINE
CT32
REAL-TIME CHEMISTRY
SYBR green
Preferentially binds double-stranded DNA
Fluorescence increases upon binding
106 copiesDNA
103 copiesDNA
Anal. Biochem. 245: 154-160; 1997 33
REAL-TIME CHEMISTRY
SYBR green
Preferentially binds double-stranded DNA
Fluorescence increases upon binding
Specific and non-specific DNA products
SOLUTION: melting curve analysis
34
Two strands of nucleic acid will melt apartd fl ill d
Slowly increase temperature (post-reaction)
MELTING CURVE ANALYSIS
and fluorescence will decrease
Function of: length of amplified targetnature of sequencepercentage G C---
35
A. Hepatitis B sAg geneC. Human -globin geneB. Combination of A and C
MELTING CURVE ANALYSIS
Anal. Biochem. 245: 154-160; 1997 36
7
A. Hepatitis B sAg geneC. Human -globin geneB. Combination of A and C
MELTING CURVE ANALYSIS
Characteristic melting peak (Tm)
Primer dimers, non-specific products withdifferent Tm; give broader peaks
Anal. Biochem. 245: 154-160; 1997 37
REAL-TIME CHEMISTRY
FRET probes
Fluorescent Resonance Energy Transfer
SYBR green
R Qprobe
gy
Means of increasing specificity
Fluorescent (reporter) and quencher dyes
38
5’ EXONUCLEASE PCR (TaqMan)
Taq polymerase also exhibits5’ exonuclease activity
39
Taq polymerase also exhibits5’ exonuclease activity
Extra, non-extending labeled hybridizationprobe (internal to target)
5’ EXONUCLEASE PCR (TaqMan)
probe (internal to target)
Primer extension displaces, cleaves probe
Specificity due to location of signal probe
fluorescence indicates PCR product
40
TARGET5’ 3’
5’ EXONUCLEASE PCR (TaqMan)
TARGET3’ 5’
41
TARGET5’ 3’
5’ EXONUCLEASE PCR (TaqMan)
TARGET3’ 5’
Denaturation 42
8
PRIMER
TARGET5’ 3’
3’ 5’
5’ EXONUCLEASE PCR (TaqMan)
PRIMER
TARGET3’ 5’
5’ 3’
Primer annealing 43
5’ 3’
PRIMER
TARGET5’ 3’
3’ 5’
5’ EXONUCLEASE PCR (TaqMan)
PRIMER
TARGET3’ 5’
5’ 3’PROBE
R Q5 3
Probe annealing 44
5’ 3’
PRIMER
TARGET5’ 3’
3’ 5’
Taql
5’ EXONUCLEASE PCR (TaqMan)
PRIMER
TARGET3’ 5’
5’ 3’PROBE
R Q5 3pol
Primer extension (polymerization) 45
5’ 3’
PRIMER
TARGET5’ 3’
3’ 5’
Taql
5’ EXONUCLEASE PCR (TaqMan)
PRIMER
TARGET3’ 5’
5’ 3’PROBE
R Q5 3pol
Primer extension (polymerization) 46
3’
PRIMER
TARGET5’ 3’
3’ 5’
Taql
5’ EXONUCLEASE PCR (TaqMan)
PRIMER
TARGET3’ 5’
5’ 3’OBE
Q3pol
Probe cleavage via 5’ exonuclease activity
R
FLUORESCENCE EMISSION
47
Q
PRIMER
TARGET5’ 3’
3’ 5’
Taql
5’ EXONUCLEASE PCR (TaqMan)
PRIMER
TARGET3’ 5’
5’ 3’pol
Primer extension (polymerization) completed
R
FLUORESCENCE EMISSION
48
9
FRET probes
SYBR green
Molecular beacons
REAL-TIME CHEMISTRY
Molecular beacons
Nat. Biotechnol. 16: 49-53; 1998 49
Hairpin oligonucleotide probe with internally-quenched fluorophore (close proximity)
Resulting hybrid morestable than stem;
MOLECULAR BEACONS
stable than stem;conformational changeforces stem apart
Fluorophore and quencher move away;fluorescence restored
Nat. Biotechnol. 16: 49-53; 1998 50
More sensitive
More specific
Can be quantitative
ADVANTAGES OF REAL-TIME PCR
Can be quantitative
Less prone to contamination
More compatible with automation
Assessment of inhibition (internal control)51
FLUORE
Analyte ofinterest
positive
BASELINE
Two colorESCENCE
Internalcontrol
TIME
BASELINE
Two-colorstain
52
FLUORE
Analyte ofinterest
Analyte ofinterest
positive negative
BASELINE
ESCENCE
Internalcontrol
Internalcontrol
TIME
BASELINE
53
FLUORE
Analyte ofinterest
Analyte ofinterest
Analyte ofinterest
positive negative unresolved
BASELINE
ESCENCE
Internalcontrol
Internalcontrol
Internalcontrol
TIME
BASELINE
54
10
55
TranscriptionTranscription
56
BASIC TENETS--I
DNA is transcribed into RNA
RNA is single-stranded molecule
Ribose (-OH at carbon #2)
O
HOCH2
2’
Three types of RNA
rRNA (ribosome structure; 5S rRNA, 16S, 23S)tRNA (brings amino acid to ribosome)mRNA (carries DNA message to ribosome)
Uracil substituted for thymine OH OH
57
Not continuous
Energy conservationInitiator, terminator regions
BASIC TENETS--II
RNA polymerase binds to promoter
Core enzymeSigma factor ( )
, g
σ58
59
POLYMERIZATIONTemplate (non-coding) DNA 3’ GTACAC 5’Complement (coding) DNA 5’ CATGTG 3’
Transcript RNA 5’ CAUGUG 3’
Transcription
No proofreading--not a big deal
Many mRNA are short-livedMany copies are made
…if RNA not functional, new one will be made
RNA nucleotides added to free 3’-OH
60
11
TERMINATION
INVERTED
3’5’
INVERTEDREPEAT
61
TERMINATION
Rho-independent termination
Rho-dependent termination
Rho-independent termination
62
FINAL PRODUCT (TRANSCRIPT)
Startcodon
Stopcodoncodon codon
Possibleregulatoryfunction
Recognitionof mRNA
at ribosome63
Diagnostic Application: Transcription mediated amplificationDiagnostic Application: Transcription‐mediated amplification
64
Reverse transcriptase-mediated formationof ds cDNA sequences containing bindingsites for T7 DNA-dependentRNA polymerase
BASIC TENETS OF TMA
RNA polymerase
Transcription
RNA transcripts re-enter cycle65
STEPS INVOLVED IN TMA
TARGETRNA
J. Clin. Virol. 25: S23-S29; 2002 66
12
STEPS INVOLVED IN TMA
TARGETRNA
PRIMER #1 POL
J. Clin. Virol. 25: S23-S29; 2002 67
STEPS INVOLVED IN TMA
TARGETRNA
PRIMER #1 POLReverse
Transcriptase
J. Clin. Virol. 25: S23-S29; 2002 68
STEPS INVOLVED IN TMA
TARGETRNA
POLTARGETcDNA
J. Clin. Virol. 25: S23-S29; 2002 69
STEPS INVOLVED IN TMA
POLTARGETcDNA
J. Clin. Virol. 25: S23-S29; 2002 70
STEPS INVOLVED IN TMA
POLTARGETcDNAPRIMER #2
J. Clin. Virol. 25: S23-S29; 2002 71
STEPS INVOLVED IN TMA
POLTARGETcDNAPRIMER #2 Polymerase
J. Clin. Virol. 25: S23-S29; 2002 72
13
STEPS INVOLVED IN TMA
POLTARGET
ds cDNATARGET POL
J. Clin. Virol. 25: S23-S29; 2002 73
STEPS INVOLVED IN TMA
POLTARGET
ds cDNATARGET POL
RNApolymerase
J. Clin. Virol. 25: S23-S29; 2002 74
STEPS INVOLVED IN TMA
ds cDNA RNApolymerase
J. Clin. Virol. 25: S23-S29; 2002 75
RNA
STEPS INVOLVED IN TMA
RNApolymerase
Transcription
RNA
J. Clin. Virol. 25: S23-S29; 2002 76
STEPS INVOLVED IN TMA
RNApolymerase
J. Clin. Virol. 25: S23-S29; 2002 77
Multiple, complex reactions simultaneouslyat one temperature (isothermal)
Rapid kinetics
POTENTIAL ADVANTAGES OF TMA
Amplify RNA targets without RNA isolationor DNase pretreatment
Rapid kinetics
“Low” risk of contamination78
14
Detection of hepatitis C in previously-negative clinical specimens
SOME LIGHT READING
Am. J. Gastroenterol. 96: 2968-2972; 2001Hepatology 32: 818-823; 2000
Multicopy rRNA target
In vitro lower limit of detection experiments
J. Clin. Microbiol. 44: 400-405; 2006J. Med. Microbiol. 54: 357-360; 2005
J. Clin. Microbiol. 35: 1369-1372; 1997 79
THE ENDStuff we’ve done
Nucleic acid hybridization
N l i id lifi ti
Liquid phase (hybridization protection)Solid phase (Southern hybridization)
In situ hybridization (& FISH)
Polymerase Chain Reaction (& real-time)Nucleic acid amplification Polymerase Chain Reaction (& real time)Transcription-mediated amplification
80
THE ENDStuff we’ve done
Nucleic acid hybridization
N l i id lifi ti
Liquid phase (hybridization protection)Solid phase (Southern hybridization)
In situ hybridization (& FISH)
Polymerase Chain Reaction (& real-time)
See you at the Dells
Nucleic acid amplification Polymerase Chain Reaction (& real time)Transcription-mediated amplification
81