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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.

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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

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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

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2

UNWIND DOUBLE HELIX

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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

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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

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Molecular Diagnostics ClassificationsMolecular Diagnostics Classifications

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Probe anneals to target of interest

NUCLEIC ACID HYBRIDIZATION

TARGET DETECTProbe

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PROBE TECHNOLOGY

More effective on colonial growth than onprimary clinical specimens

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3

Amplify target of interest prior to detection

NUCLEIC ACID AMPLIFICATIONTESTING (NAAT)

TARGET

Primer

PrimerDETECTAMPLIFY

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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)

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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)

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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

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Surface area to volume ratio

HOW DOES THIS HAPPEN?!?!

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Surface area to volume ratio (increased)

HOW DOES THIS HAPPEN?!?!

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Real-time detection

Surface area to volume ratio

HOW DOES THIS HAPPEN?!?!

No target control

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Real-time detection

Surface area to volume ratio

HOW DOES THIS HAPPEN?!?!

BASELINE

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6

Real-time detection

Surface area to volume ratio

HOW DOES THIS HAPPEN?!?!

BASELINE

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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

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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---

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A. Hepatitis B sAg geneC. Human -globin geneB. Combination of A and C

MELTING CURVE ANALYSIS

Anal. Biochem. 245: 154-160; 1997 36

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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

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5’ EXONUCLEASE PCR (TaqMan)

Taq polymerase also exhibits5’ exonuclease activity

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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

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TARGET5’ 3’

5’ EXONUCLEASE PCR (TaqMan)

TARGET3’ 5’

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TARGET5’ 3’

5’ EXONUCLEASE PCR (TaqMan)

TARGET3’ 5’

Denaturation 42

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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

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Q

PRIMER

TARGET5’ 3’

3’ 5’

Taql

5’ EXONUCLEASE PCR (TaqMan)

PRIMER

TARGET3’ 5’

5’ 3’pol

Primer extension (polymerization) completed

R

FLUORESCENCE EMISSION

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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

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FLUORE

Analyte ofinterest

Analyte ofinterest

positive negative

BASELINE

ESCENCE

Internalcontrol

Internalcontrol

TIME

BASELINE

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FLUORE

Analyte ofinterest

Analyte ofinterest

Analyte ofinterest

positive negative unresolved

BASELINE

ESCENCE

Internalcontrol

Internalcontrol

Internalcontrol

TIME

BASELINE

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TranscriptionTranscription

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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

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Not continuous

Energy conservationInitiator, terminator regions

BASIC TENETS--II

RNA polymerase binds to promoter

Core enzymeSigma factor ( )

, g

σ58

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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

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TERMINATION

INVERTED

3’5’

INVERTEDREPEAT

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TERMINATION

Rho-independent termination

Rho-dependent termination

Rho-independent termination

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FINAL PRODUCT (TRANSCRIPT)

Startcodon

Stopcodoncodon codon

Possibleregulatoryfunction

Recognitionof mRNA

at ribosome63

Diagnostic Application: Transcription mediated amplificationDiagnostic Application:  Transcription‐mediated amplification

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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

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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

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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

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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

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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

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