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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
Review
3
COMPONENTS
DNA
Phosphate
Pentose(deoxyribose)
Base(thymine)
O
O-
PO
-O
-
OH H
O
HOCH2
N
O
CH3
NH
O
2’
4
COMPOSITE
OH H
O
CH2
O H
O
CH2
O
O-
POO
-
O
POO
-
NN
N
N
N H
H
Adenine
N
O
N
N
H
H H
Cytosine
5
N
N
N
N
N H
H
N
N
N
N
O
N HH
H
N
O
CH3
NH
O
N
O
N
NH
H
Adenine
Guanine
Thymine
Cytosine
H
Hketo group amino group
HYDROGEN BONDING
6
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-
POO
-
NN
N
N
N H
H
Adenine
Cytosine
O
POO
-
N
O
N
N
H
H H
OH H
O
CH2
O-
5’
5’
3’
3’
DNApolymerase( ) x 3
9
Molecular Diagnostics Classifications
10
Probe anneals to target of interest
TARGET DETECTProbe
NUCLEIC ACID HYBRIDIZATION
11
PROBE TECHNOLOGY
More effective on colonial growth than onprimary clinical specimens
12
Amplify target of interest prior to detection
TARGET
Primer
PrimerDETECTAMPLIFY
NUCLEIC ACID AMPLIFICATIONTESTING (NAAT)
13
ANALYTICAL SENSITIVITY
Manual of Clinical Microbiology, 6th edition; 1995 14
Denature(95°C, 5 min)
Anneal/hybridize(30°C, 2 min)
Repeat 19 times;add Klenow each time
Klenow extension(30°C, 2 min)
PROTOCOL (Mullis et al.)
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)
~40 cycles
Extension (72°C)
“OPTIMIZED” PCR PROTOCOL
17
Elapsed time: 3 hours
18
Elapsed time: 4 hours
19
Elapsed time: 4.5 hours
20
Elapsed time: 6.5 hours
21
Elapsed time: 7 hours
22
Alternatively…
Elapsed time: 5.5 hours
23
OLD
SCHOOL 24
25
Diagnostic Application: Real-time PCR
Elapsed time: ~1 hour
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
No target control
HOW DOES THIS HAPPEN?!?!
29
BASELINE
Real-time detection
Surface area to volume ratio
HOW DOES THIS HAPPEN?!?!
30
BASELINE
Real-time detection
Surface area to volume ratio
HOW DOES THIS HAPPEN?!?!
31
BASELINE
CT
Cycle threshold (CT)Real-time detection
Surface area to volume ratio
HOW DOES THIS HAPPEN?!?!
32
106 copiesDNA
103 copiesDNA
REAL-TIME CHEMISTRY
Anal. Biochem. 245: 154-160; 1997
SYBR green
Preferentially binds double-stranded DNA
Fluorescence increases upon binding
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 apartand fluorescence will decrease
Slowly increase temperature (post-reaction)
Function of: length of amplified targetnature of sequencepercentage G C ---
MELTING CURVE ANALYSIS
35
A. Hepatitis B sAg geneC. Human -globin geneB. Combination of A and C
Anal. Biochem. 245: 154-160; 1997
MELTING CURVE ANALYSIS
36
Characteristic melting peak (Tm)
Primer dimers, non-specific products withdifferent Tm; give broader peaks
A. Hepatitis B sAg geneC. Human -globin geneB. Combination of A and C
Anal. Biochem. 245: 154-160; 1997
MELTING CURVE ANALYSIS
37
R Qprobe
REAL-TIME CHEMISTRY
FRET probes
Fluorescent Resonance Energy Transfer
Means of increasing specificity
Fluorescent (reporter) and quencher dyes
SYBR green
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)
Primer extension displaces, cleaves probe
Specificity due to location of signal probe
fluorescence indicates PCR product
5’ EXONUCLEASE PCR (TaqMan)
40
TARGET3’ 5’
TARGET5’ 3’
5’ EXONUCLEASE PCR (TaqMan)
41
TARGET3’ 5’
TARGET5’ 3’
Denaturation
5’ EXONUCLEASE PCR (TaqMan)
42
PRIMER
TARGET3’ 5’
5’ 3’
PRIMER
TARGET5’ 3’
3’ 5’
Primer annealing
5’ EXONUCLEASE PCR (TaqMan)
43
PRIMER
TARGET3’ 5’
5’ 3’PROBE
R Q5’ 3’
PRIMER
TARGET5’ 3’
3’ 5’
Probe annealing
5’ EXONUCLEASE PCR (TaqMan)
44
PRIMER
TARGET3’ 5’
5’ 3’PROBE
R Q5’ 3’
PRIMER
TARGET5’ 3’
3’ 5’
Taqpol
Primer extension (polymerization)
5’ EXONUCLEASE PCR (TaqMan)
45
PRIMER
TARGET3’ 5’
5’ 3’PROBE
R Q5’ 3’
PRIMER
TARGET5’ 3’
3’ 5’
Taqpol
Primer extension (polymerization)
5’ EXONUCLEASE PCR (TaqMan)
46
PRIMER
TARGET3’ 5’
5’ 3’OBE
Q3’
PRIMER
TARGET5’ 3’
3’ 5’
Taqpol
Probe cleavage via 5’ exonuclease activity
R
FLUORESCENCE EMISSION
5’ EXONUCLEASE PCR (TaqMan)
47
PRIMER
TARGET3’ 5’
5’ 3’
Q
PRIMER
TARGET5’ 3’
3’ 5’
Taqpol
Primer extension (polymerization) completed
R
FLUORESCENCE EMISSION
5’ EXONUCLEASE PCR (TaqMan)
48
FRET probes
SYBR green
Molecular beacons
REAL-TIME CHEMISTRY
Nat. Biotechnol. 16: 49-53; 1998 49
Hairpin oligonucleotide probe with internally-quenched fluorophore (close proximity)
Resulting hybrid morestable than stem;conformational changeforces stem apart
Fluorophore and quencher move away;fluorescence restored
MOLECULAR BEACONS
Nat. Biotechnol. 16: 49-53; 1998 50
More sensitive
More specific
Can be quantitative
Less prone to contamination
More compatible with automation
Assessment of inhibition (internal control)
ADVANTAGES OF REAL-TIME PCR
51
FLUORESCENCE
Analyte ofinterest
Internalcontrol
TIME
positive
BASELINE
BASELINE
Two-colorstain
52
FLUORESCENCE
Analyte ofinterest
Analyte ofinterest
Internalcontrol
Internalcontrol
TIME
positive negative
BASELINE
BASELINE
53
FLUORESCENCE
Analyte ofinterest
Analyte ofinterest
Internalcontrol
Internalcontrol
Analyte ofinterest
Internalcontrol
TIME
positive negative unresolved
BASELINE
BASELINE
54
55
Transcription
56
BASIC TENETS--I
Three types of RNA
rRNA (ribosome structure; 5S rRNA, 16S, 23S)tRNA (brings amino acid to ribosome)mRNA (carries DNA message to ribosome)
DNA is transcribed into RNA
RNA is single-stranded molecule
Ribose (-OH at carbon #2)Uracil substituted for thymine OH OH
O
HOCH2
2’
57
RNA polymerase binds to promoter
Core enzymeSigma factor ( )
Not continuous
Energy conservationInitiator, terminator regions
BASIC TENETS--II
σ58
59
POLYMERIZATION
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
Template (non-coding) DNA 3’ GTACAC 5’Complement (coding) DNA 5’ CATGTG 3’
Transcript RNA 5’ CAUGUG 3’
Transcription
60
TERMINATION
3’5’
INVERTEDREPEAT
61
TERMINATION
Rho-dependent termination
Rho-independent termination
62
FINAL PRODUCT (TRANSCRIPT)
Startcodon
Stopcodon
Possibleregulatoryfunction
Recognitionof mRNA
at ribosome63
Diagnostic Application: Transcription-mediated amplification
64
Reverse transcriptase-mediated formationof ds cDNA sequences containing bindingsites for T7 DNA-dependentRNA polymerase
Transcription
RNA transcripts re-enter cycle
BASIC TENETS OF TMA
65
TARGETRNA
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 66
TARGETRNA
PRIMER #1 POL
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 67
TARGETRNA
PRIMER #1 POLReverse
Transcriptase
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 68
TARGETRNA
POLTARGETcDNA
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 69
POLTARGETcDNA
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 70
POLTARGETcDNAPRIMER #2
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 71
POLTARGETcDNAPRIMER #2 Polymerase
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 72
POLTARGET
ds cDNATARGET POL
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 73
POLTARGET
ds cDNATARGET POL
RNApolymerase
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 74
POL
TARGET
ds cDNA
TARGET
POL
RNApolymerase
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 75
POL
TARGET
TARGET
POL
RNApolymerase
TARGET
TARGET
Transcription
RNA
RNA
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 76
TARGET
TARGET
RNApolymerase
TARGETTARGET
TARGETTARGET
TARGETTARGET
TARGETTARGET
TARGETTARGET
TARGETTARGET
TARGET
TARGETTARGET
TARGETTARGET
TARGETTARGET
TARGETTARGET
TARGETTARGET
TARGETTARGET
TARGETTARGET
TARGET
STEPS INVOLVED IN TMA
J. Clin. Virol. 25: S23-S29; 2002 77
Multiple, complex reactions simultaneouslyat one temperature (isothermal)
Amplify RNA targets without RNA isolationor DNase pretreatment
Rapid kinetics
POTENTIAL ADVANTAGES OF TMA
“Low” risk of contamination78
Detection of hepatitis C in previously-negative clinical specimens
Multicopy rRNA target
In vitro lower limit of detection experiments
SOME LIGHT READING
Am. J. Gastroenterol. 96: 2968-2972; 2001Hepatology 32: 818-823; 2000
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
Nucleic acid amplification
Liquid phase (hybridization protection)Solid phase (Southern hybridization)
In situ hybridization (& FISH)
Polymerase Chain Reaction (& real-time)Transcription-mediated amplification
80
THE END
See you at the Dells
Stuff we’ve done
Nucleic acid hybridization
Nucleic acid amplification
Liquid phase (hybridization protection)Solid phase (Southern hybridization)
In situ hybridization (& FISH)
Polymerase Chain Reaction (& real-time)Transcription-mediated amplification
81
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