Project Title:Project Title:

Project Manager: Project Manager:

Prof. of Molecular PathologyProf. of Molecular PathologyDirector of Molecular Biology Research UnitDirector of Molecular Biology Research Unit

AssiutAssiut University, University, AssiutAssiut, Egypt, Egypt

Fourth Cycle Funded Project, 2005Fourth Cycle Funded Project, 2005

Prof. of Molecular PathologyProf. of Molecular PathologyDirector of Molecular Biology Research UnitDirector of Molecular Biology Research Unit

AssiutAssiut University, University, AssiutAssiut, Egypt, Egypt20052005

11--Genetic mappingGenetic mapping22--Genetic polymorphismGenetic polymorphism33--Detection of mutationsDetection of mutations44--Molecular virologyMolecular virology55--Microbiology and human diseases Microbiology and human diseases

(molecular diagnosis )(molecular diagnosis )66-- Forensic medicine (DNA fingerprinting )Forensic medicine (DNA fingerprinting )77-- PhylogeneticsPhylogenetics88-- analysis of gene expression by detection of analysis of gene expression by detection of

mRNA mRNA (RT(RT-- PCR )PCR )

APPLICATIONS OF PCRAPPLICATIONS OF PCR

This lecture has been This lecture has been on three on three

topics: topics:

I- Molecular diagnosis of viral diseases.Example: HCV

II- Confirming the diagnosis of zoonoticbacterial diseases.Examples: a- Salmonellosis b- Listeriosis c- E. coliO157:H7

III- Phylogenetic studies by DNA fingerprinting. Examples: a- E. coli b- Fusiforms c- Trichophyton

Scope of this lectureScope of this lecture

Use of PCR in Molecular VirologyUse of PCR in Molecular Virology

Work carried out routinely in our unit Work carried out routinely in our unit includes:includes:

A)A)-- Qualitative analysis of gene Qualitative analysis of gene expression of HCV by RTexpression of HCV by RT-- PCR.PCR.

B)B)-- Quantitative analysis of HCV by Quantitative analysis of HCV by RealReal-- Time PCR.Time PCR.

USESUSESDetect or quantify the expression of Detect or quantify the expression of messages from small amounts of RNAmessages from small amounts of RNA

•• Analysis of differential gene Analysis of differential gene expression or clone expression or clone cDNAcDNA without without constructing a constructing a cDNAcDNA librarylibrary

•• More sensitive and easier to perform More sensitive and easier to perform than other RNA analysis techniquesthan other RNA analysis techniques

REVERSE TRANSCRIPTREVERSE TRANSCRIPT--PCRPCR(RT(RT-- PCR)PCR)

IDEAIDEARTRT-- PCR combines PCR combines cDNAcDNA synthesis from RNA synthesis from RNA

templates with PCR to provide a rapid, templates with PCR to provide a rapid, method for method for analysinganalysing gene expressiongene expression

11--Template RNA ( total or poly ( A ) selected RNA )Template RNA ( total or poly ( A ) selected RNA )

22--Primers of RTPrimers of RT-- PCRPCR ::

( For first strand ( For first strand cDNAcDNA synthesis )synthesis )

Random PrimersRandom Primers::

-- Less specificLess specific

-- Anneal to multiple sites along the entire Anneal to multiple sites along the entire short partial length short partial length cDNAcDNA..

OligoOligo ((dtdt ) primers) primers::

-- More specific than random primersMore specific than random primers

-- Hybridize to the poly ( A ) tail found at the 3Hybridize to the poly ( A ) tail found at the 3,,

eukaryotic mRNAeukaryotic mRNA

GeneGene-- specific primersspecific primers::

-- Most specificMost specific

-- Antisence oligonucleotideAntisence oligonucleotide that hybridize to that hybridize to sequencesequence

ComponentsComponents

33-- Reverse Reverse TranscriptasesTranscriptases: :

-- Moloney murineMoloney murine leukemia leukemia

-- Avian Avian myeloplastosis myeloplastosis virus ( AMV ) virus ( AMV )

-- Superscript 11 RTSuperscript 11 RT

-- ThermoscriptThermoscript RT ( the best )RT ( the best )

Components Components

RTRT-- PCRPCR

TwoTwo-- step RTstep RT-- PCR: PCR: --cDNAcDNA synthesis synthesis performed first in RT performed first in RT bufferbuffer

One step RT One step RT PCR : reverse PCR : reverse and PCR take place sequentially in and PCR take place sequentially in tubetube

SUCCESSFUL RTSUCCESSFUL RT--PCR MEANSPCR MEANS::

--High sensitivity i.e. getting High sensitivity i.e. getting product from small samplesproduct from small samples

-- High specificity i.e. High specificity i.e. amplification of the desired amplification of the desired

TYPES OF RTTYPES OF RT-- PCRPCR

RTRT-- PCR for HCVPCR for HCV

P C R p ositive an d n egative H C V p a tien ts u sin g R T - P C R

Three principle fields:Three principle fields:

11-- Absolute quantification of genes ( i.e. Absolute quantification of genes ( i.e. viruses, bacteria, fungi, etc ).viruses, bacteria, fungi, etc ).

22-- Gene expression ( i.e. cytokines, growth Gene expression ( i.e. cytokines, growth factors, transcription factors, etc. ).factors, transcription factors, etc. ).

33-- Allelic discrimination ( detection of single Allelic discrimination ( detection of single nucleotide polymorphism, SNP ).nucleotide polymorphism, SNP ).

RealReal-- Time PCRTime PCRPractical Applications.Practical Applications.

*Salmonellosis or salmonella infection is one of the most important bacterial diseases affecting a wide spectrum of hosts including human and birds.

*Poultry has been recognized as an importance source of such zoonoticpathogens .

**SalmonellosisSalmonellosis or salmonella infection or salmonella infection is one of the most important bacterial is one of the most important bacterial diseases affecting a wide spectrum of diseases affecting a wide spectrum of hosts including human and birds. hosts including human and birds.

*Poultry has been recognized as an *Poultry has been recognized as an importance source of such importance source of such zoonoticzoonoticpathogens pathogens ..

Molecular Diagnosis of Bacterial Infections

Molecular Diagnosis of Bacterial Infections

In our work,First:First:

**PCR technique has been adapted to give accurate PCR technique has been adapted to give accurate diagnosis of diagnosis of SalmonellosisSalmonellosis in chicken either from in chicken either from culture or field samples by using specific primers. culture or field samples by using specific primers.

Second:Second:

** Determination of the sensitivity and specificity of the Determination of the sensitivity and specificity of the PCR assay in comparison to conventional methods PCR assay in comparison to conventional methods for diagnosis of avian for diagnosis of avian salmonellosissalmonellosis has been has been evaluated.evaluated.

1600 bp

800 bp

100 bp

1600 bp

800 bp

100 bp -300 bp-300 bp

Agarose gel electrophoresis of PCR products following amplificationof DNAs of different Salmonella species.

Lane (M) : 100 bp ladder.Lane(1-9) : Amplified fragments of 300 and 1600 bp from

different DNA of Salmonella isolates extracted from cultures.

Lane (10) : Amplified fragments of 300 bp of Salmonella typhimurium (Reference strain).

Lane (11) : Negative control (No template DNA).

Lane (M) : 100 bp ladder.Lane(1-9) : Amplified fragments of 300 and 1600 bp from

different DNA of Salmonella isolates extracted from cultures.

Lane (10) : Amplified fragments of 300 bp of Salmonella typhimurium (Reference strain).

Lane (11) : Negative control (No template DNA).

-300 bp-300 bp

1600 bp

800 bp

100 bp

1600 bp

800 bp

100 bp

Agarose gel electrophoresis of PCR products following amplification of DNA extracted from field samples.

Lane (M) : 100bp ladder.Lane (1, 3, 6, 7, 9) : Represent negative field samples.Lane (2, 4): Represent PCR products from faecal samples.

(1600bp amplified products).Lane (5, 8) : Represent PCR products from faecal samples.

(800bp amplified products).Lane (10) : Negative control. (No template DNA). Lane (11) : Reference strain. (300bp amplified products).

Lane (M) : 100bp ladder.Lane (1, 3, 6, 7, 9) : Represent negative field samples.Lane (2, 4): Represent PCR products from faecal samples.

(1600bp amplified products).Lane (5, 8) : Represent PCR products from faecal samples.

(800bp amplified products).Lane (10) : Negative control. (No template DNA). Lane (11) : Reference strain. (300bp amplified products).

CONCLUSION

11--The results confirmed the efficiency and The results confirmed the efficiency and success of PCR technique in detecting success of PCR technique in detecting Salmonella DNA from laboratory cultures.Salmonella DNA from laboratory cultures.

22--PCR amplification was more sensitive than PCR amplification was more sensitive than other serologic techniques in detecting other serologic techniques in detecting salmonella species from these cultures.salmonella species from these cultures.

33--PCR amplification was also more reliable PCR amplification was also more reliable and specific for detection of Salmonella and specific for detection of Salmonella DNA directly from DNA directly from faecalfaecal samples than samples than other conventional methods.other conventional methods.

44--PCR amplification assay successfully PCR amplification assay successfully allowed the detection of salmonella DNA in allowed the detection of salmonella DNA in 46 out of 72 46 out of 72 faecalfaecal samples tested.samples tested.

Detection of Detection of zoonoticzoonotic pathogens from milk and milk productspathogens from milk and milk productsNested PCR has been used to detect Nested PCR has been used to detect ListeriaListeria monocytogensmonocytogens using using

specific primers for specific primers for ListeriolysinListeriolysin O gene .O gene .

R e s u l t s : T h r e e posit ive cases are diagnosed among 220 s a m p l e s o f m i l k , cheese and butter.

Conclusion: PCR has proved very sensitive method for diagnosing serious food- borne pathogens that could not b e d e t e c t e d b y s e r o l o g i c a l m e t h o d s .

*In this work, PCR has been used to detect E. coli O157: H7 in meat products using specific primers to hlyA gene.* PCR confirmed the diagnosis of only one case (lane 1) isolated from 75 beefburger samples of 400 bp that could not be diagnosed serologically.

Conclusion: PCR is a rapid and automated method with proven high specificity and sensitivity in detecting microbial pathogens.

It has faster attainment of confirmed results.

It has higher sample throughput than traditional culture methods.

Diagnosis of Diagnosis of zoonoticzoonotic pathogens in pathogens in meat productsmeat products

IDEA :IDEA :

•• Certain sequences of DNA occur many Certain sequences of DNA occur many

at different positions along the at different positions along the

•• These positions vary considerably, These positions vary considerably,

give rise to a polymorphism that is give rise to a polymorphism that is

the individual, specie or strain.the individual, specie or strain.

•• The most common technique is:The most common technique is:

GENETIC FINGERPRINTINGGENETIC FINGERPRINTING

Random Amplified Random Amplified DNA DNA -- PCR ( RAPD PCR ( RAPD PCR )PCR )

APPLICATIONSAPPLICATIONS::

-- Gene mappingGene mapping

-- Detection of strain diversityDetection of strain diversity

-- Population analysisPopulation analysis

-- Demonstration of Demonstration of phylogeneticphylogenetic and taxonomic and taxonomic

-- Detection of polymorphisms at a number of Detection of polymorphisms at a number of

•• RAPD analysis is a technique for rapidly RAPD analysis is a technique for rapidly

genomic genomic polmorphismspolmorphisms utilizing a single utilizing a single

oligonucleotideoligonucleotide primer of arbitrary primer of arbitrary

polymerasepolymerase chain reaction ( PCR ). chain reaction ( PCR ).

•• The PCR reaction is carried out under low The PCR reaction is carried out under low

conditions to generate a reproducible conditions to generate a reproducible

specific products that are subsequently specific products that are subsequently

gel electrophoresis.gel electrophoresis.

IDEA OF RAPD IDEA OF RAPD -- PCRPCR

--Thermostable TaqThermostable Taq DNA DNA

--DNTPsDNTPs

--BufferBuffer

--BSABSA

RAPDRAPD-- PCR Beads containPCR Beads contain

Reagents added:Reagents added:

-- Template DNATemplate DNA : :

genomic from any organism genomic from any organism

Should be pure, high qualityShould be pure, high quality

-- Primers:Primers:

•• Single Single oligonucleotideoligonucleotide of arbitrary of arbitrary

•• 7 7 15 bases in length15 bases in length

•• G/C content should be at least 60 % G/C content should be at least 60 %

•• Different primer concentrations give Different primer concentrations give

of bandsof bands

RAPD RAPD -- PCRPCR

PCR reaction:PCR reaction:

1 cycle at 95 1 cycle at 95 ooCC for 5 minutesfor 5 minutes

45 cycles at 95 45 cycles at 95 ooCC for 1 min, 36 for 1 min, 36

min.and 72 min.and 72 ooCC for 2 min.for 2 min.

RAPD RAPD -- PCRPCR

Gel analysis:Gel analysis:

2 % 2 % agaroseagarose using 1X TBE or TAEusing 1X TBE or TAE

o.5 o.5 ugug/ ml / ml ethidiumethidium bromide bromide

Electrophoresis at 150 V for 1.5 Electrophoresis at 150 V for 1.5

Analysis of RAPD markers ( Analysis of RAPD markers (

or cluster analysis ): Using Scan or cluster analysis ): Using Scan

software software

RAPD RAPD -- PCRPCR

* In this work, 30 E. * In this work, 30 E. colicoli strains were isolated from strains were isolated from diseased broilers and layer chickens.diseased broilers and layer chickens.* The isolates were serologically identified using* The isolates were serologically identified usingOO-- antigen.antigen.* Some strains could not be serologically typed.* Some strains could not be serologically typed.

Phylogenetic Studies by Random Amplified Polymorphic DNA- PCR

(RAPD- PCR)

Aim: Studying the genetic diversity among the groups of E. coli strains previously identified by serological t echn iques , bes ide t he un t ypab le s t r a i ns .

RAPD- PCR

Agarose gel showing RAPD profiles that are polymorphic between 15 E.coli isolates using

random primer 5.

PhylogeneticPhylogenetic Tree or Cluster Analysis of Thirty Tree or Cluster Analysis of Thirty Different E. Different E. coli Serotypescoli Serotypes Isolated From Broilers and Isolated From Broilers and

layer Chickenslayer Chickens•The isolated strains were typed into 6 major clonal clusters.

•Inter-serotypic variation is observed among each cluster.

•The untypable strains are grouped into 3 different clusters that have genetic relationship to other E. coli serotypes..

•The strain O157 is of zoonotic importance and is genetically related to O11 in cluster 6.

Conclusion: PCR- based fingerprinting methods are simple and powerful tools for genetic characterization of the organism and may contribute to better understanding of the epidemiology of this Pathogen.

Other example for RAPDOther example for RAPD-- PCRPCRStudying the genetic diversity of different strains of Studying the genetic diversity of different strains of

Genus Genus FusiformFusiform (A) and Genus (A) and Genus TrichophytonTrichophyton (B).(B).A B

Agarose gel electrophoresis showing different RAPD markers between the different strains of Genus Fusiform(A) and Genus Trichophyton (B) that indicates variable DNA homology among different strains of each genus.

Cluster analysis of RAPDCluster analysis of RAPD-- PCR markersPCR markersfor different strains of for different strains of FusiformFusiform (A)(A) and and TrichophytonTrichophyton (B).(B).

The phylogenetic trees of both A and B demonstrate genetic variations among the tested strains based on their DNA composition.

A B

Another PCRAnother PCR--based techniquebased technique

Restriction Fragment Length Restriction Fragment Length polymorphism (RFLP)polymorphism (RFLP)

What are the restriction enzymes What are the restriction enzymes

( Restriction ( Restriction EndonucleasesEndonucleases ))

Restriction enzymes bind specifically Restriction enzymes bind specifically double strandeddouble stranded-- DNA at specific sites DNA at specific sites adjacent a particular sequence known as adjacent a particular sequence known as Recognition SequenceRecognition Sequence

RRSTRICTION FRAGMENT LENGTH RRSTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSISPOLYMORPHISM ANALYSIS

( RFLP )( RFLP )

Eg. HpaI and HpaII - Haemophilus parainfluenzaefirst and second R.E. isolated from above

Capitalised letter = genus

lowercase letters = first 2 letters of the species name

Roman numerals = order of characterisation

Eg. HpaI and HpaII - Haemophilus parainfluenzaefirst and second R.E. isolated from above

Capitalised letter = genus

lowercase letters = first 2 letters of the species name

Roman numerals = order of characterisation

Restriction enzymes: NamingRestriction enzymes: Naming

EnzymeEnzyme Recognition sequence Recognition sequence EcoEcoRI RI GAATTCGAATTCHinHindIII dIII AAGCTTAAGCTTBamBamHI HI GGATCCGGATCCEcoEcoRV RV GATATCGATATC

Recognition sequences are usually 4Recognition sequences are usually 4--8 8 base pairs in length and are usually base pairs in length and are usually palindromicpalindromic

ExamplesExamples

R.E.s are classified into 3 groups

Type I & III: Bind to RS but cleave DNA randomly thus not used in molecular cloning.

Type II: (a) Cleaves specific DNA sequences at or close to RS allowing manipulation of DNA

(b) Primary tools of molecular biologists

(c) Allow cloning and purification of defined DNA fragments

R.E.s are classified into 3 groups

Type I & III: Bind to RS but cleave DNA randomly thus not used in molecular cloning.

Type II: (a) Cleaves specific DNA sequences at or close to RS allowing manipulation of DNA

(b) Primary tools of molecular biologists

(c) Allow cloning and purification of defined DNA fragments

R.E.s are classified into 3 groups

Type II Restriction enzymesType II Restriction enzymes recognize recognize

that could be:that could be:

Four nucleotidesFour nucleotides in length ( Four base in length ( Four base

Five nucleotidesFive nucleotides in length ( Five base in length ( Five base

Six nucleotidesSix nucleotides in length ( Six base in length ( Six base

-- Some enzymes cleave both strands in Some enzymes cleave both strands in

of the recognition sequence, of the recognition sequence,

fragments of DNA that carry fragments of DNA that carry BLUNT BLUNT

-- Others cleave DNA asymmetrically, Others cleave DNA asymmetrically,

fragments of DNA that carry fragments of DNA that carry PROTRUDING PROTRUDING

STICKY ENDS.STICKY ENDS.

The RS for most are palindromes(base sequence same on both strands of DNA when read 5’to 3’)

The location of cleavage differs between enzymes yielding differentsets of cuts:

1) some cleave at the axis of symmetry and generate blunt endseg. Hae III

5’ . . . . . . G G C C . . . . . . 3’3’ . . . . . . C C G G . . . . . . 5’

5’ . . . . . . G G C C . . . . . . 3’3’ . . . . . . C C G G . . . . . . 5’

The RS for most are palindromes(base sequence same on both strands of DNA when read 5’to 3’)

The location of cleavage differs between enzymes yielding differentsets of cuts:

1) some cleave at the axis of symmetry and generate blunt endseg. Hae III

5’ . . . . . . G G C C . . . . . . 3’3’ . . . . . . C C G G . . . . . . 5’

5’ . . . . . . G G C C . . . . . . 3’3’ . . . . . . C C G G . . . . . . 5’

Type II R.E.

A closer lookA closer look……. . EcoEcoRVRV

5’….ACTGTACGATATCGCTA….3’5’….ACTGTACGATATCGCTA….3’3’….TGACATGCTATAGCGAT….5’3’….TGACATGCTATAGCGAT….5’

A closer lookA closer look……. . EcoEcoRVRV

5’….ACTGTACGAT ATCGCTA….3’5’….ACTGTACGAT ATCGCTA….3’3’….TGACATGCTA TAGCGAT….5’3’….TGACATGCTA TAGCGAT….5’

A closer lookA closer look……. . EcoEcoRVRV

5’….ACTGTACGAT ATCGCTA….3’5’….ACTGTACGAT ATCGCTA….3’3’….TGACATGCTA TAGCGAT….5’3’….TGACATGCTA TAGCGAT….5’

“blunt ends”-can bind with otherDNA molecules withblunt ends

“blunt ends”-can bind with otherDNA molecules withblunt ends

2) Most REs make staggered cuts in the opposite strand creating sticky ends for example:

(a) Cleaves each strand of DNA on the 5’ side of the axis of symmetryfragments carry protruding cohesive 5’ terminieg Eco R1:

2) Most REs make staggered cuts in the opposite strand creating sticky ends for example:

(a) Cleaves each strand of DNA on the 5’ side of the axis of symmetryfragments carry protruding cohesive 5’ terminieg Eco R1:

5’ . . . . . . G A A T T C . . . . . . 3’3’ . . . . . . C T T A A G . . . . . . 5’

5’ . . . . . . G A A T T C . . . . . . 3’3’. . . . . . C T T A A G . . . . . . 5’

5’ overhang 5’ overhang

5’ . . . . . . G A A T T C . . . . . . 3’3’ . . . . . . C T T A A G . . . . . . 5’

5’ . . . . . . G A A T T C . . . . . . 3’3’. . . . . . C T T A A G . . . . . . 5’

5’ overhang 5’ overhang

A closer lookA closer look……. . BamBamHIHI

BamHI

5’….ACTGTACGGATCCGCTA….3’5’….ACTGTACGGATCCGCTA….3’3’….TGACATGCCTAGGCGAT….5’3’….TGACATGCCTAGGCGAT….5’

A closer lookA closer look……. . BamBamHIHI

5’….ACTGTACGGATCCGCTA….3’5’….ACTGTACGGATCCGCTA….3’3’….TGACATGCCTAGGCGAT….5’3’….TGACATGCCTAGGCGAT….5’

BamHI

A closer lookA closer look……. . BamBamHIHI

5’….ACTGTACG GATCCGCTA….3’5’….ACTGTACG GATCCGCTA….3’3’….TGACATGCCTAG GCGAT….5’3’….TGACATGCCTAG GCGAT….5’

A closer lookA closer look……. . BamBamHIHIGATCCGCTA….3’

GCGAT….5’

GCTA….3’GCGAT….5’5’….ACTGTACG

3’….TGACATGCCTAG5’….ACTGTACG

3’….TGACATGCCTAG

GATCC

“sticky ends”-can bind with other DNA molecules with the same overhangs

“sticky ends”-can bind with other DNA molecules with the same overhangs

Compatible endsCompatible ends

GATCCGCTA….3’GATCCGCTA….3’GCGAT….5’GCGAT….5’5’….ACTGTACG5’….ACTGTACG

3’….TGACATGCCTAG3’….TGACATGCCTAG

GATCTGCTA….3’GATCTGCTA….3’ACGAT….5’ACGAT….5’

5’….ACTGTACA5’….ACTGTACA3’….TGACATGTCTAG3’….TGACATGTCTAG

Compatible endsCompatible ends

5’….ACTGTACAGATCCGCTA….3’5’….ACTGTACAGATCCGCTA….3’3’….TGACATGTCTAGGCGAT….5’3’….TGACATGTCTAGGCGAT….5’

(b) Cleaves each strand on the 3’ side of the axis of symmetry –yieldsfragments of DNA with protruding cohesive 3’ termini eg Pst1

(b) Cleaves each strand on the 3’ side of the axis of symmetry –yieldsfragments of DNA with protruding cohesive 3’ termini eg Pst1

5’ . . . . . . C T G C A G . . . . . . 3’3’ . . . . . . G A C G T C . . . . . . 5’

5’ . . . . . . C T G C A G . . . . . . 3’3’ . . . . . . G A C G T C . . . . . . 5’

3’overhang 3’overhang

5’ . . . . . . C T G C A G . . . . . . 3’3’ . . . . . . G A C G T C . . . . . . 5’

5’ . . . . . . C T G C A G . . . . . . 3’3’ . . . . . . G A C G T C . . . . . . 5’

3’overhang 3’overhang

RFLPRFLP