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

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Molecular Diagnostics Classifications

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Probe anneals to target of interest

TARGET DETECTProbe

NUCLEIC ACID HYBRIDIZATION

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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?!?!

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106 copiesDNA

103 copiesDNA

REAL-TIME CHEMISTRY

Anal. Biochem. 245: 154-160; 1997

SYBR green

Preferentially binds double-stranded DNA

Fluorescence increases upon binding

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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 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)

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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

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Transcription

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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

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TERMINATION

3’5’

INVERTEDREPEAT

61

TERMINATION

Rho-dependent termination

Rho-independent termination

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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

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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

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