MOLECULAR BIOLOGY TECHNIQUES USED IN ZOONOTIC DISEASE DIAGNOSIS

Dr. Satyanarayan Prusti.

INTRODUCTIONINTRODUCTION Zoonotic pathogens cause diseases and death both in Zoonotic pathogens cause diseases and death both in

human & animals which ultimately leads to man power human & animals which ultimately leads to man power and economic loss of the country.and economic loss of the country.

Traditional diagnostic methods identify a pathogen Traditional diagnostic methods identify a pathogen based on its based on its phenotypephenotype..

The correct assessment of a clinical isolate The correct assessment of a clinical isolate takes more takes more time.time.

Faster and simpler methods of diagnosis is of great Faster and simpler methods of diagnosis is of great advantage.advantage.

That is why molecular biology technique is the first and That is why molecular biology technique is the first and foremost choice .foremost choice .

MOLECULAR BIOLOGYMOLECULAR BIOLOGY

The branch of biology that deals with The branch of biology that deals with the formation, structure, and the formation, structure, and function of macromolecules essential function of macromolecules essential to life, such as nucleic acids and to life, such as nucleic acids and proteins, and especially with their proteins, and especially with their role in cell replication and the role in cell replication and the transmission of genetic information.transmission of genetic information.

MOLECULAR BIOLOGY MOLECULAR BIOLOGY TECHNIQUETECHNIQUE

The biological technique in which The biological technique in which macromolecules like nucleic acid and macromolecules like nucleic acid and proteins are used is known as Molecular proteins are used is known as Molecular Biology Technique.Biology Technique.

WHY GO FOR MOLECULAR WAY?WHY GO FOR MOLECULAR WAY?

Traditional methods pose Traditional methods pose several challengesseveral challenges

Growth of Growth of fastidious fastidious pathogenspathogens Maintenance of Maintenance of viabilityviability Delay in Delay in cultivationcultivation Non-culturabilityNon-culturability of certain of certain

organisms.organisms. HazardousHazardous to propagate in lab. to propagate in lab. CostCost versus versus clinical utilityclinical utility..

Advantage of Molecular MethodsAdvantage of Molecular Methods

Aid in Aid in faster diagnosis faster diagnosis of diseasesof diseases.. Increased Increased sensitivity and specificity.sensitivity and specificity. Rapid detection of pathogenRapid detection of pathogen than than

conventional methods.conventional methods. Identification of Identification of epidemiologically important epidemiologically important

strains .strains . Decrease the Decrease the man powerman power needneed for pathogen for pathogen

detection.detection. Give Give rapid answers rapid answers to treatment options in to treatment options in life life

threatening infections.threatening infections. Adapted to Adapted to instrumentation.instrumentation.

MOLECULAR METHOD

Protein method Nucleic acid method

There are about 30 different types of molecular biological tests—some protein, some nucleic acid method. The challenge is comparing them and determining which is more reliable, specific, or sensitive.

The biggest molecular diagnostics is nucleic acid testing. New processes have already improved this method.

.

NUCLEIC ACID METHOD

Hybridization assay

amplification assays

• Nucleic acid probes & hybridization.

• Hybrid capture

• DNA Microarray

• Polymerase chain reaction

•LigaseChainReaction

• NASBA

• SDA

•RAPD

•RFLP

•Gene sequencing

HYBRIDIZATION HYBRIDIZATION ASSAYASSAY

NUCLEIC ACID PROBE AND NUCLEIC ACID PROBE AND HYBRIDIZATIONHYBRIDIZATION

Release of nucleic Release of nucleic acid from specimenacid from specimen

Denaturation (dsDNA)Denaturation (dsDNA)

Hybridization with Hybridization with probeprobe

Detection of hybridDetection of hybrid

Hybridization :Hybridization :

Ability of Ability of 2 nucleic acid strands 2 nucleic acid strands that have that have complementarycomplementary base sequences base sequences to specifically to specifically bond with each other & form a bond with each other & form a double stranded double stranded molecule (duplex or hybrid)molecule (duplex or hybrid)

Probe:Probe:

Nucleic acid strand Nucleic acid strand from an organism of from an organism of known known identityidentity. Usually probes are 100-100000 base . Usually probes are 100-100000 base long.long.

Conjugated probe :Conjugated probe : ( radio active, enzyme etc) ( radio active, enzyme etc)

Target:Target:

Nucleic acid Nucleic acid strand of the organism to be strand of the organism to be detecteddetected or identified or identified

Duplex:Duplex: (positive hybridization) (positive hybridization)

Duplex can be Duplex can be DNA-DNADNA-DNA, , DNA-RNADNA-RNA & even & even RNA-RNA-RNA RNA depending upon the design of the assaydepending upon the design of the assay

HYBRIDIZATION STEPS & COMPONENTSHYBRIDIZATION STEPS & COMPONENTS

1. Production & labeling of single- 1. Production & labeling of single- stranded probe nucleic acidstranded probe nucleic acid

2. Preparation of single stranded target 2. Preparation of single stranded target nucleic acidnucleic acid

3. Mixture & hybridization of target & 3. Mixture & hybridization of target & probe nucleic acidprobe nucleic acid

4. Detection of hybridization4. Detection of hybridization

I.PRODUCTION OF SINGLE STRAND PROBE I.PRODUCTION OF SINGLE STRAND PROBE NUCLEIC ACIDNUCLEIC ACID

Depends on the sequence of intended target Depends on the sequence of intended target nucleic acid (ie. intended to use)nucleic acid (ie. intended to use)

Probes are commercially synthesized and Probes are commercially synthesized and labeled with reporter molecule.labeled with reporter molecule.

The user need to supply the manufacturer with The user need to supply the manufacturer with the desired nucleotide base sequencethe desired nucleotide base sequence

II. Preparation of target nucleic acidII. Preparation of target nucleic acid Target nucleic acid must be single Target nucleic acid must be single

stranded & its base sequence integrity stranded & its base sequence integrity should be intactshould be intact

Target preparation stepsTarget preparation steps

1. 1. Enzymatic/chemical Enzymatic/chemical destruction of thedestruction of the

microbial envelope to release the target microbial envelope to release the target nucleic acidnucleic acid

2. 2. StabilizationStabilization of target nucleic acid to of target nucleic acid to preserve structural integritypreserve structural integrity

3. If target is DNA, 3. If target is DNA, denaturationdenaturation into single into single strand ( generally heated to 94 C)strand ( generally heated to 94 C)

III. MIXTURE & HYBRIDIZATION OFIII. MIXTURE & HYBRIDIZATION OF

TARGET & PROBETARGET & PROBE

EnvironmentEnvironment in which probe & target are in which probe & target are brought together brought together isis importantimportant

Hybridization stringency is most affected byHybridization stringency is most affected by

A. A. Salt concentrations Salt concentrations in hybridization in hybridization bufferbuffer

B. B. TemperatureTemperature

C. Concentration of C. Concentration of destabilizing destabilizing agentsagents

NUCLEIC ACID PROBE & HYBRIDIZATION

IV. DETECTION OF HYBRIDIZATIONIV. DETECTION OF HYBRIDIZATION

Depends on the reporter molecule used for Depends on the reporter molecule used for labelling probe nucleic acidlabelling probe nucleic acid

3 main reporter system used are3 main reporter system used are

1. 1. RadioactiveRadioactive reporter reporter

2. 2. Biotin-avidinBiotin-avidin reporter reporter

3. 3. ChemiluminiscentChemiluminiscent reporter reporter

HYBRID CAPTURE TECHNIQUE:HYBRID CAPTURE TECHNIQUE:

Solution formatSolution format

Solid support formatSolid support format

Southern hybridizationsSouthern hybridizations Northern hybridizationsNorthern hybridizations Dot Blot hybridizationDot Blot hybridization In situ hybridizationsIn situ hybridizations

SOLUTION HYBRIDIZATION FORMATSOLUTION HYBRIDIZATION FORMAT

SOLID SUPPORT HYBRIDIZATION FORMATSOLID SUPPORT HYBRIDIZATION FORMAT

1.Southern blot hybridization1.Southern blot hybridization

2.2. Northern blot hybridizationsNorthern blot hybridizations

3.Dot Blot hybridization3.Dot Blot hybridization

4.Insitu hybridization4.Insitu hybridization

SOUTHERN BLOT HYBRIDIZATIONSOUTHERN BLOT HYBRIDIZATION

DNA fragments are separated by DNA fragments are separated by gel electrophoresisgel electrophoresis,, usually an agarose gel.usually an agarose gel.

Restriction fragments on the gel appears as a Restriction fragments on the gel appears as a smearsmear rather than discrete bands. rather than discrete bands.

DenatureDenature the fragments by incubation with NaOH. the fragments by incubation with NaOH.

TransferredTransferred to a membrane which is a sheet of to a membrane which is a sheet of special special blotting paper.blotting paper.

The DNA fragments will retain the The DNA fragments will retain the same patternsame pattern of separation they had on the gel.of separation they had on the gel.

The blot is The blot is incubatedincubated with many copies of with many copies of labeled labeled

probe probe which are single-stranded.which are single-stranded.

This probe will form base pairs with its This probe will form base pairs with its complementarycomplementary

DNA DNA sequence to form a sequence to form a double-strandeddouble-stranded DNA molecule. DNA molecule.

The location of the probe is revealed by The location of the probe is revealed by incubating it incubating it

with a colorless substrate with a colorless substrate that the attached that the attached enzyme enzyme

converts to a colored productconverts to a colored product

If the probe was labeled with If the probe was labeled with radioactivityradioactivity,,

it can expose it can expose X-ray film directlyX-ray film directly. .

SOUTHERN HYBRIDIZATIONSOUTHERN HYBRIDIZATION

Southern blot/ DNA blotSouthern blot/ DNA blot

NORTHERN BLOT HYBRIDIZATION

NORTHERN BLOT HYBRIDIZATION

DOT BLOT HYBRIDIZATIONDOT BLOT HYBRIDIZATION Detection of a Detection of a given sequence of DNA/ RNAgiven sequence of DNA/ RNA, , not not

fractionatedfractionated (not subjected to gel electrophoresis). (not subjected to gel electrophoresis).

Method-Method- DNA (or RNA) DNA (or RNA) from different samples are from different samples are transferred onto a transferred onto a nitrocellulose filter nitrocellulose filter in form of in form of dotdot..

The DNA is first The DNA is first denatureddenatured and then the filter is and then the filter is backed at 80ºC backed at 80ºC to fix to fix the DNA firmly onto the filter.the DNA firmly onto the filter.

Appropriate Appropriate radioactive single stranded DNA probe radioactive single stranded DNA probe is is added added

Hybridization with radioactive probes is detected by Hybridization with radioactive probes is detected by autoradiographyautoradiography..

the the intensity of dotintensity of dot in the autoradiograph in the autoradiograph corresponds the corresponds the extent to which DNA or RNA extent to which DNA or RNA is is represented in the sample. represented in the sample.

INSITU HYBRIDIZATIONINSITU HYBRIDIZATION

Allows a pathogen to be Allows a pathogen to be identifiedidentified within the within the context of the pathologic context of the pathologic lesionlesion being being

producedproduced Uses Uses patient cells or tissues as the patient cells or tissues as the

solid support phasesolid support phase Combines the Combines the power of molecular power of molecular

diagnosisdiagnosis withwith the additional the additional information that information that histopathology histopathology examinationexamination can provide. can provide.

INSITU HYBRIDIZATIONINSITU HYBRIDIZATION

Molecular Molecular pathologypathology

1.1. FISH (fluorescent FISH (fluorescent labeled probe)labeled probe)

2.2. CISH CISH (chemiluminiscent (chemiluminiscent probe)probe)

FISH Broken Down Into Five Steps:

1)  Identify Probe – Prepare short sequences of DNA which are complementary to the target DNA sequences.

2)  Label Probe - Fluorescent dye(Fluorescein is an example of a fluorescent marker) is used to label the probe.

3)  Denature Chromosome and Probe - First, denature the chromosomes and then probe by way of heat or pH. 

4)  Hybridization - Add the denatured chromosomes and the denatured probe to a microscope slide.  Allow the probe to hybridize to its complementary site. 

5)  Analysis - Finally wash away the excess probe and observe the probe hybridization by using a fluorescence microscope.

CHEMILUMINISCENCE INSITU CHEMILUMINISCENCE INSITU HYBRIDIZATIONHYBRIDIZATION

Is performed using probes that are detected using Is performed using probes that are detected using enzymes enzymes with with their their appropriate chemiluminescent substrates. appropriate chemiluminescent substrates.

The The luminescent signal luminescent signal from the hybrid formation is detected, from the hybrid formation is detected, analyzed and measured with analyzed and measured with a high performance low light level a high performance low light level imaging apparatus imaging apparatus connected to an connected to an optical microscope optical microscope and to a and to a personal personal computercomputer for quantitative image analysis. for quantitative image analysis.

This provides the This provides the estimation and quantification estimation and quantification of nucleic acids of nucleic acids present in tissue samples or cellular smearspresent in tissue samples or cellular smears

STEPS OF CISHSTEPS OF CISH 1)  1)  Identify Probe- Identify Probe- – Prepare short sequences of DNA which – Prepare short sequences of DNA which

are complementary to the are complementary to the target DNA sequences.target DNA sequences. 2)  2)  Label Probe-Label Probe-The probe is labeled with The probe is labeled with chemical hapten chemical hapten

like digoxigenin.like digoxigenin. 3)   3)   Denature Chromosome and Probe Denature Chromosome and Probe - First, denature the - First, denature the

chromosomes by way of heat or pH.  Then denature the chromosomes by way of heat or pH.  Then denature the probe via heat or pH.probe via heat or pH.

4)  4)  HybridizationHybridization - Add the denatured chromosomes and the - Add the denatured chromosomes and the denatured probe to a denatured probe to a microscope slidemicroscope slide.  Allow the probe to .  Allow the probe to hybridize to its complementary site.  hybridize to its complementary site. 

5) 5) Addition of chemiluminescent- Addition of chemiluminescent- Anti-digoxigenin Fab Anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate phosphate substrate for alkaline phosphatase are added to for alkaline phosphatase are added to the slide.the slide.

6)  6)  AnalysisAnalysis – After washing the unhybridized probes, – After washing the unhybridized probes, luminescent signalluminescent signal from the hybrid formation was detected, from the hybrid formation was detected, analyzed, and measured with analyzed, and measured with luminograph apparatus luminograph apparatus connected to an optical microscope connected to an optical microscope and to a and to a personal personal computer computer for quantitative image analysis.for quantitative image analysis.

Figure - Chemiluminescence in situ hybridization revealing increasing concentrations target DNA in a sample.

DNA MICROARRAYDNA MICROARRAY

The core The core principleprinciple behind microarrays is behind microarrays is hybridizationhybridization between between two DNA two DNA strands. The strands. The property of property of complementary nucleic acid sequences nucleic acid sequences to specifically pair with each other by forming to specifically pair with each other by forming hydrogen bonds between complementary between complementary nucleotide base pairs..

A high number of complementary base pairs in a A high number of complementary base pairs in a nucleotide sequence means tighter nucleotide sequence means tighter non-non-covalent bonding between the two strands.bonding between the two strands.

Fluorescently labeled targetFluorescently labeled target sequences that sequences that bindbind to to a probe sequence generate a a probe sequence generate a signalsignal that depends on that depends on the the strength of the hybridization strength of the hybridization determined by the determined by the number of paired bases.number of paired bases.

DNA MICROARRAYDNA MICROARRAY DNA DNA oligonucleotide probes are attached oligonucleotide probes are attached

to the to the solid supportsolid support..(glass,nylon,silocon,ploypropylene etc).(glass,nylon,silocon,ploypropylene etc).

Then unknown DNA undergo PCR to Then unknown DNA undergo PCR to produce more number of target DNA produce more number of target DNA fragments.fragments.

Labeled the Labeled the target DNA target DNA with with fluorescent fluorescent dyedye..

Add this labeled DNAAdd this labeled DNA on to the known on to the known probe DNA.probe DNA.

Allow this hybridization to complete. Allow this hybridization to complete. The The hybridized probe hybridized probe can be detected in can be detected in

sensitive sensitive detection system.detection system.

DNA MICROARRAYDNA MICROARRAY

DNA MICROARRAYDNA MICROARRAY

Fixed probes

labeled target(sample)

Different Features(eg bind different genes)

Fully complementary strands bind strongly

AMPLIFICATION ASSAY

.PCR

• LCR

• NASBA

• SDA

•RAPD

•RFLP

•GENE SEQUENCING

WHY AMPLIFICATION??WHY AMPLIFICATION?? If If sufficient target nucleic acid sufficient target nucleic acid is not is not

present in the reaction, hybridization can present in the reaction, hybridization can give false negative results.give false negative results.

To To circumventcircumvent this, nucleic acid this, nucleic acid amplification is usedamplification is used

How to amplify the nucleic acid?How to amplify the nucleic acid?

By allowing By allowing repeated replication repeated replication of target of target nucleic acid by the specific primer.nucleic acid by the specific primer.

POLYMERASE CHAIN REACTION(PCR)POLYMERASE CHAIN REACTION(PCR)

CombinesCombines the principles of the principles of complementarycomplementary nucleic acid nucleic acid hybridizationhybridization with those of with those of nucleic acid nucleic acid replicationreplication that are applied that are applied repeatedly through numerous cycles for repeatedly through numerous cycles for generation thousands to millions copies of generation thousands to millions copies of a particular DNA sequence.a particular DNA sequence.

The method relies on The method relies on thermal cycling, , consisting of cycles of repeated heating consisting of cycles of repeated heating and cooling of the reaction for and cooling of the reaction for DNA melting and and enzymatic replication of the DNA. of the DNA.

PCR REQUIREMENTPCR REQUIREMENT DNA templateDNA template that contains the DNA region (target) to that contains the DNA region (target) to

be amplified.be amplified. primers that are that are complementary to the to the 3' (three prime) (three prime)

ends of each of the ends of each of the sense and anti-sense strand of the strand of the DNA target.DNA target.

Taq polymeraseTaq polymerase or another DNA polymerase with a or another DNA polymerase with a temperature optimum at around 70 °C.temperature optimum at around 70 °C.

DeoxynucleotideDeoxynucleotide the building blocks from which the the building blocks from which the DNA polymerases synthesizes a new DNA strand.DNA polymerases synthesizes a new DNA strand.

Buffer solution,Buffer solution, providing a suitable chemical providing a suitable chemical environment for optimum activity and stability of the environment for optimum activity and stability of the DNA polymerase.DNA polymerase.

Divalent cations,Divalent cations, magnesium or manganese ions magnesium or manganese ions Monovalent cationMonovalent cation potassium ions. potassium ions.

THERMAL CYCLERTHERMAL CYCLER To maintain continuous reaction To maintain continuous reaction

cycles, programmable thermal cycles, programmable thermal cyclers are usedcyclers are used

POLYMERASE CHAIN POLYMERASE CHAIN REACTION(PCR)REACTION(PCR)

Basic steps:Basic steps:

Denaturation of the target (dsDNA)Denaturation of the target (dsDNA)

Annealing of primersAnnealing of primers

Extension of primer-target DuplexExtension of primer-target Duplex

Detection of PCR productsDetection of PCR products

DENATURATION OF NUCLEIC ACIDDENATURATION OF NUCLEIC ACID

Target nucleic acid is added to the Target nucleic acid is added to the reaction mix that contains all necessary reaction mix that contains all necessary components of PCR to occur (primers, components of PCR to occur (primers, covalent ions, buffers, enzymes etc)covalent ions, buffers, enzymes etc)

Denaturation of dsDNA to a single strand Denaturation of dsDNA to a single strand is accomplished by is accomplished by heating to 94 Cheating to 94 C

PRIMER ANNEALINGPRIMER ANNEALING PrimersPrimers are short single sequences of are short single sequences of

nucleotides (18-24 nucleotides)Selected nucleotides (18-24 nucleotides)Selected specifically specifically to flank the target sequence of to flank the target sequence of interest.interest.

When the primer pair is mixed with the When the primer pair is mixed with the denatured target DNA, denatured target DNA, one primer anneals one primer anneals to a specific site at 3’ endto a specific site at 3’ end of the target of the target strand, while the strand, while the other primer anneals to a other primer anneals to a specific site at the 3’ end of the otherspecific site at the 3’ end of the other, , complementary target strandcomplementary target strand

Once the duplexes are formed Once the duplexes are formed (40-60c), (40-60c), the last step in cycle which mimics the the last step in cycle which mimics the DNA replication process, beginsDNA replication process, begins

DENATURATION &PRIMER ANNEALING

EXTENSION OF PRIMEREXTENSION OF PRIMER

TAQ POLYMERASETAQ POLYMERASE is the enzyme commonly used is the enzyme commonly used for primer extension,for primer extension, which occurs at which occurs at 72 C72 C

It can function efficiently at elevated It can function efficiently at elevated temperature & withstand the denaturing temperature & withstand the denaturing temperature of 94 C through several cycles.temperature of 94 C through several cycles.

Annealing of primers to target sequence provides Annealing of primers to target sequence provides the the necessary template formatnecessary template format that allows that allows DNA DNA polymerase to add nucleotides to 3’ end of each polymerase to add nucleotides to 3’ end of each primerprimer & produce & produce by extension a sequenceby extension a sequence complementary to target sequencecomplementary to target sequence

For each target sequenceFor each target sequence originally present in the originally present in the PCR mixture, PCR mixture, 2 double stranded fragments2 double stranded fragments containing the target sequence are containing the target sequence are produced produced after one cycleafter one cycle

At the beginning of the At the beginning of the second cycle of PCR,second cycle of PCR, denaturation then produces 4 templatesdenaturation then produces 4 templates to which to which the the primers will anneal.primers will anneal.

Following extension at the end of the second Following extension at the end of the second cycle, there will be cycle, there will be 4 double stranded fragments 4 double stranded fragments containing target nucleic acidcontaining target nucleic acid

After After 30 to 40 cycles, 1030 to 40 cycles, 107 7 to 10to 108 8 targettarget copies will copies will be present in the reaction mixturebe present in the reaction mixture

POLYMERASE CHAIN POLYMERASE CHAIN REACTION(PCR)REACTION(PCR)

Seq

uen

tial ro

un

ds

DETECTION OF PCR PRODUCTDETECTION OF PCR PRODUCT

(Post amplification analysis)(Post amplification analysis)

-Gel electrophoresis is the most common method -Any of the basic methods previously described

for detecting hybridization can also be adapted

GEL ELECTROPHORESISGEL ELECTROPHORESIS A portion of PCR mixture after amplification is A portion of PCR mixture after amplification is

subjected to subjected to gel electrophoresisgel electrophoresis After electrophoresis, the gel is stained with After electrophoresis, the gel is stained with

ethidium bromideethidium bromide to to visualize the ampliconvisualize the amplicon Using Using molecular weight size markersmolecular weight size markers, the presence , the presence

of amplicons of appropriate size is confirmed.of amplicons of appropriate size is confirmed.

GEL ELECTROPHORESISGEL ELECTROPHORESIS

GEL ELECTROPHORESISGEL ELECTROPHORESIS

POLYMERASE CHAIN REACTIONPOLYMERASE CHAIN REACTION

Types of PCRTypes of PCR■ Nested PCRNested PCR Broad range PCRBroad range PCR Multiplex PCRMultiplex PCR RT - PCRRT - PCR Real Time PCRReal Time PCR

NESTED PCRNESTED PCR

Two primers added Two primers added sequentiallysequentially

First amplicon serves as a First amplicon serves as a target for second amplificationtarget for second amplification

STEPS OF NESTED PCR

Step One: The DNA target template is bound by the first set of primers shown in blue. The primers may bind to alternative, similar primer binding sites which give multiple products however only one of these PCR products give the intended sequence (multiple products not shown). Step Two: PCR products from the first PCR reaction are subjected to a second PCR run however with a second new set of primers shown in red.As these primers are NESTED within the first PCR product, they make it very unlikely that non-specifically amplified PCR product would contain binding sites for both sets of primers. This nested PCR amplification ensures that the PCR product from the second PCR amplification has little or no contamination from non-specifically amplified PCR products from alternative primer target sequences.

BROAD RANGE PCRBROAD RANGE PCR

Use of broad range Use of broad range specificity primersspecificity primers

Advantage:Advantage: Primers target Primers target a larger group of a larger group of microorganismsmicroorganisms

Disadvantage:Disadvantage: detection detection of phylogenetically related of phylogenetically related organisms but not those organisms but not those in the group of interestin the group of interest

MULTIPLEX PCRMULTIPLEX PCR

Multiple primersMultiple primers Each primer can target a different Each primer can target a different

organismorganism Used to detect:Used to detect:

• Viral agents causing encephalitisViral agents causing encephalitis(HSV, Enteroviruses, West nile (HSV, Enteroviruses, West nile

viruses)viruses) Bacterial agents causing meningitisBacterial agents causing meningitis(N. meningitidis ,M. tuberculosis etc)(N. meningitidis ,M. tuberculosis etc) Enteric pathogens (Salmonella - Enteric pathogens (Salmonella -

campylobacter ,Shigella,E.colicampylobacter ,Shigella,E.coli

REVERSE TRANSCRIPTASE PCRREVERSE TRANSCRIPTASE PCR

Target - RNATarget - RNA Reverse transcriptaseReverse transcriptase makes a makes a cDNAcDNA

copy of targetcopy of target DNA polymerase makes a DNA polymerase makes a dsDNAdsDNA Routine PCR Routine PCR technique is donetechnique is done Useful for detecting RNA virusesUseful for detecting RNA viruses

REAL TIME PCRREAL TIME PCR

Real-time polymerase chain reactionReal-time polymerase chain reaction, also called , also called quantitative real time quantitative real time polymerase chain reaction (Q-polymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chain reaction, is a PCR/qPCR) or kinetic polymerase chain reaction, is a laboratory technique based on the PCR, which is used laboratory technique based on the PCR, which is used to to amplify and simultaneously quantify a targeted DNA amplify and simultaneously quantify a targeted DNA molecule.molecule.

The procedure follows the general principle of The procedure follows the general principle of polymerase chain reaction; its key feature is that the polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in amplified DNA is detected as the reaction progresses in real time.real time.

The products are detected and measured in the The products are detected and measured in the real-real-time PCR thermocycler,time PCR thermocycler,

METHODS OF REALTIME PCRMETHODS OF REALTIME PCR Two different methods are used.Two different methods are used.

Method -1:Method -1:--Real-time PCR using double-stranded DNA dyes.Real-time PCR using double-stranded DNA dyes.

In this method in addition to the all the PCR mix dsDNA dyes such as In this method in addition to the all the PCR mix dsDNA dyes such as SYBR Green is added.SYBR Green is added.

This dye will bind to all This dye will bind to all dsDNA PCR productsdsDNA PCR products, including nonspecific PCR , including nonspecific PCR products.products.

Method -2:-Method -2:-Fluorescent reporter probe methodFluorescent reporter probe method

In this method in addition to the all the PCR mix In this method in addition to the all the PCR mix Fluorescent probes Fluorescent probes are are added.added.

During the annealing stage of the PCR both During the annealing stage of the PCR both probe and primers probe and primers anneal to anneal to the DNA target.the DNA target.

Polymerization Polymerization of a new DNA strand is initiated from the primers, and of a new DNA strand is initiated from the primers, and once the once the polymerase reaches the probepolymerase reaches the probe, its 5'-3-exonuclease , its 5'-3-exonuclease degrades degrades the probethe probe, physically separating the fluorescent reporter resulting in an , physically separating the fluorescent reporter resulting in an increase in fluorescenceincrease in fluorescence. .

Real Time PCRReal Time PCR

LIGASE CHAIN REACTIONLIGASE CHAIN REACTION

The The ligase chain reactionligase chain reaction (LCR) is a method of (LCR) is a method of DNA amplification. While the better-known PCR DNA amplification. While the better-known PCR carries out the amplification by polymerizing carries out the amplification by polymerizing nucleotides, LCR instead amplifies the nucleic nucleotides, LCR instead amplifies the nucleic acid used as the probe. For each of the two DNA acid used as the probe. For each of the two DNA strands, strands, two partial probes are ligated to form the two partial probes are ligated to form the actual oneactual one; thus, LCR uses two enzymes: a DNA ; thus, LCR uses two enzymes: a DNA polymerase and a DNA ligase. Each cycle results polymerase and a DNA ligase. Each cycle results in a doubling of the target nucleic acid molecule.in a doubling of the target nucleic acid molecule.

LCR is a LCR is a good diagnostic potential good diagnostic potential but major but major limitation is the limitation is the expensiveexpensive requirement of requirement of two two pair of deoxyoligomers.pair of deoxyoligomers.

STEPS OF LIGASE CHAIN STEPS OF LIGASE CHAIN REACTIONREACTION

DenaturationDenaturation of target DNA at 94c. of target DNA at 94c. Annealing Annealing of two pairs of synthetic of two pairs of synthetic

deoxyoligomeres at40-60c.deoxyoligomeres at40-60c. Members of each pair bind in such a way that Members of each pair bind in such a way that

they are they are immediately adjacent immediately adjacent and completely and completely cover the target sequence cover the target sequence on both separated on both separated DNA strands.DNA strands.

The oligomeres of each pair is then The oligomeres of each pair is then joinedjoined by a by a thermostable DNA thermostable DNA ligase.ligase.

This This doublesdoubles the target DNA molecules and the target DNA molecules and complete the complete the first LCR cyclefirst LCR cycle

The The second cyclesecond cycle is initiated by is initiated by denaturationdenaturation. .

LIGASE CHAIN REACTION

NUCLEIC ACID SEQUENCE BASED NUCLEIC ACID SEQUENCE BASED AMPLIFICATION (NASBA)AMPLIFICATION (NASBA)

NASBA is an ingenious method NASBA is an ingenious method basedbased on on retroviral replication retroviral replication for for amplification of amplification of RNARNA(or DNA with (or DNA with modification to this method).modification to this method).

This is more complex than PCR.But This is more complex than PCR.But its does its does notnot require require thermal cyclingthermal cycling..

It proceeds rapidly and isothermally It proceeds rapidly and isothermally at at 37c.37c.

STEPS OF NASBASTEPS OF NASBAThe viral The viral RNA strands RNA strands are represented as the sense strand are represented as the sense strand present in the original samples.present in the original samples.

Primer P1 Primer P1 binds to the RNA and is elongated by reverse binds to the RNA and is elongated by reverse transcriptase (AMV-RT). The RNA strand of the yielded transcriptase (AMV-RT). The RNA strand of the yielded DNA : DNA : RNA hybrid RNA hybrid is hydrolyzed by is hydrolyzed by RNase H.RNase H.

After the binding of P1, After the binding of P1, primer P2 primer P2 can also bind. Primer P2 is can also bind. Primer P2 is then elongated by AMV – RT, yielding then elongated by AMV – RT, yielding a double-stranded DNA a double-stranded DNA molecule. molecule.

Primer P1 is designed in such a manner that when it forms a Primer P1 is designed in such a manner that when it forms a double- stranded DNA, it codes for a double- stranded DNA, it codes for a T7 RNA polymerse T7 RNA polymerse Promoter site. This helps in generating antisense RNA copies Promoter site. This helps in generating antisense RNA copies using a DNA template.using a DNA template.

The new copies of DNA are generated using RNA. The The new copies of DNA are generated using RNA. The process is same as followed for sense strand. Here in this process is same as followed for sense strand. Here in this case, P2 will bind first.case, P2 will bind first.

NASBANASBA

STRAND DISPLACEMENT STRAND DISPLACEMENT AMPLIFICATION(SDA)AMPLIFICATION(SDA)

This process consists of This process consists of twotwo phase-phase-Target generation Target generation and second is and second is amplificationamplification..

This process requires This process requires two primer pairs two primer pairs (B1,S1 and B2,S2)(B1,S1 and B2,S2) and and two enzymes two enzymes pairs( DNA polymerase and a restriction pairs( DNA polymerase and a restriction enzyme) ,enzyme) ,dATP and dAMPdATP and dAMP..

This process is This process is efficient efficient for for small targets small targets of less than 200 bp.of less than 200 bp.

STEPS OF SDASTEPS OF SDA DenaturationDenaturation of the DNA and attachment of the of the DNA and attachment of the

primers.primers. Elongation Elongation of of B1B1 and and B2B2 primer to produce the primer to produce the

target DNA.target DNA. Primer Primer S1 and S2(having nick able site) S1 and S2(having nick able site) binds with binds with

the target DNA and elongation takes place.the target DNA and elongation takes place. Incorporation of Incorporation of dAMPdAMP into the target DNA at the into the target DNA at the

nick able site nick able site .. Nicking Nicking of the DNA strand(produced from primer s1 of the DNA strand(produced from primer s1

and s2) takes place and that DNA strands are and s2) takes place and that DNA strands are separate out.separate out.

DNA polymerase DNA polymerase again act at the nick able site and again act at the nick able site and extends that strand again and the cycle continues.extends that strand again and the cycle continues.

RANDOM AMPLIFIED POLYMORPHIC DNARANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) (RAPD)

It is a type of PCR reaction, but the segments of It is a type of PCR reaction, but the segments of DNA that are DNA that are amplified are random.amplified are random.

The scientist performing RAPD creates several The scientist performing RAPD creates several arbitrary, arbitrary, short primers short primers (8-12 nucleotides), then (8-12 nucleotides), then proceeds with the PCR using a large template of proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify.genomic DNA, hoping that fragments will amplify.

By resolving the resulting patterns, a By resolving the resulting patterns, a semi-uniquesemi-unique profile can be gleaned in profile can be gleaned in gel electrophoresisgel electrophoresis..

No knowledge of the DNA sequence No knowledge of the DNA sequence for the for the targeted gene is required, as the primers will bind targeted gene is required, as the primers will bind somewheresomewhere in the sequence, but it is not certain in the sequence, but it is not certain exactly where.exactly where.

Molecular characterization of isolates Molecular characterization of isolates using RAPD fingerprintingusing RAPD fingerprinting

RAPD fingerprints of RAPD fingerprints of reference strainsreference strains

RAPD fingerprints of local isolates.    RAPD fingerprints of local isolates.    Patterns matching with that of reference Patterns matching with that of reference strains indicated strains indicated

RESTRICTION FRAGMENT LENGTH POLYMORPHISMRESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP(RFLP))

Restriction enzymes Restriction enzymes are used to are used to cut DNA cut DNA into fragments (called restriction fragment into fragments (called restriction fragment length polymorphism) which may be length polymorphism) which may be characteristic at strain level.characteristic at strain level.

These fragments are then separated by These fragments are then separated by agarose agarose gel electrophoresisgel electrophoresis, forming , forming characteristic characteristic banding patternsbanding patterns..

Identification of the organism can then be Identification of the organism can then be established by established by comparingcomparing the pattern of the pattern of unknown organism with that of unknown organism with that of known(reference) organismknown(reference) organism

RFLPRFLP: Restriction Fragment Length : Restriction Fragment Length PolymorphismPolymorphism

sample

GENE SEQUENCINGGENE SEQUENCING Sequence of nucleotide Sequence of nucleotide of the of the target gene target gene is is

used for identification of organism.used for identification of organism. Dideoxy nucleotides Dideoxy nucleotides are added in the PCR are added in the PCR

mixture in addition to themixture in addition to the fluorescent labeled fluorescent labeled nucleotidesnucleotides. .

This Dideoxy nucleotide when added to the DNA This Dideoxy nucleotide when added to the DNA process of process of elongation stops elongation stops and denaturation and denaturation takes place.takes place.

So in this way So in this way many copy of DNA fragments many copy of DNA fragments of of DNA produced.DNA produced.

The sequence of nucleotides can be known by The sequence of nucleotides can be known by using using automated DNA sequencer automated DNA sequencer machine which machine which uses uses capillary electrophoresis and laser printercapillary electrophoresis and laser printer..

Gene sequence

CONCLUSIONCONCLUSION

The molecular methods are a The molecular methods are a promising promising alternativealternative that can that can substitute or complement substitute or complement the the current reference method used in disease current reference method used in disease diagnosis.diagnosis.

More suitable and reliable results can be achieved More suitable and reliable results can be achieved in terms of speed and precision and can deter in terms of speed and precision and can deter and enumerate specifically viable organism.and enumerate specifically viable organism.

Sample preparation problem, contamination, Sample preparation problem, contamination, entrenched attitude slow but cannot stop entrenched attitude slow but cannot stop adoption of the molecular method of diagnosis.adoption of the molecular method of diagnosis.

The man who removes a The man who removes a mountain begins by carrying mountain begins by carrying

away small stonesaway small stones