MOLECULAR BIOLOGY TECHNIQUES USED IN ZOONOTIC DISEASE

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Zoonotic pathogens cause diseases and death both in human & animals which ultimately leads to man power and economic loss of the country. Traditional diagnostic methods identify a pathogen based on …

Zoonotic pathogens cause diseases and death both in human & animals which ultimately leads to man power and economic loss of the country. Traditional diagnostic methods identify a pathogen based on its phenotype.
The correct assessment of a clinical isolate takes more time. Faster and simpler methods of diagnosis is of great advantage. That is why molecular biology technique is the first and foremost choice .

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  • How x ray film is placed ?
  • Denaturation-94 c Annealing 40-60 c Elongatin-72c,Taq polymerase,dATP. dGTP, dCTP, dTTP
  • NESTED PCR

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  • 1. MOLECULAR BIOLOGY TECHNIQUES USED IN ZOONOTIC DISEASE DIAGNOSIS Dr. Satyanarayan Prusti.
  • 2. INTRODUCTION
    • Zoonotic pathogens cause diseases and death both in human & animals which ultimately leads to man power and economic loss of the country.
    • Traditional diagnostic methods identify a pathogen based on its phenotype .
    • The correct assessment of a clinical isolate takes more time.
    • Faster and simpler methods of diagnosis is of great advantage.
    • That is why molecular biology technique is the first and foremost choice .
  • 3. MOLECULAR BIOLOGY
    • The branch of biology that deals with the formation, structure, and function of macromolecules essential to life, such as nucleic acids and proteins, and especially with their role in cell replication and the transmission of genetic information.
  • 4. MOLECULAR BIOLOGY TECHNIQUE
    • The biological technique in which macromolecules like nucleic acid and proteins are used is known as Molecular Biology Technique.
  • 5. WHY GO FOR MOLECULAR WAY?
    • Traditional methods pose several challenges
    • Growth of fastidious pathogens
    • Maintenance of viability
    • Delay in cultivation
    • Non-culturability of certain organisms.
    • Hazardous to propagate in lab.
    • Cost versus clinical utility .
  • 6. Advantage of Molecular Methods
    • Aid in faster diagnosis of diseases .
    • Increased sensitivity and specificity.
    • Rapid detection of pathogen than conventional methods.
    • Identification of epidemiologically important strains .
    • Decrease the man power need for pathogen detection.
    • Give rapid answers to treatment options in life threatening infections.
    • Adapted to instrumentation.
  • 7. 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.
  • 8. . 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
  • 9. HYBRIDIZATION ASSAY
  • 10. NUCLEIC ACID PROBE AND HYBRIDIZATION
    • Release of nucleic acid from specimen
    • Denaturation (dsDNA)
    • Hybridization with probe
    • Detection of hybrid
  • 11.
    • Hybridization :
    • Ability of 2 nucleic acid strands that have complementary base sequences to specifically bond with each other & form a double stranded molecule (duplex or hybrid)
    • Probe:
    • Nucleic acid strand from an organism of known identity . Usually probes are 100-100000 base long.
    • Conjugated probe : ( radio active, enzyme etc)
    • Target:
    • Nucleic acid strand of the organism to be detected or identified
    • Duplex: (positive hybridization)
    • Duplex can be DNA-DNA , DNA-RNA & even RNA-RNA depending upon the design of the assay
  • 12.
    • HYBRIDIZATION STEPS & COMPONENTS
    • 1. Production & labeling of single- stranded probe nucleic acid
    • 2. Preparation of single stranded target nucleic acid
    • 3. Mixture & hybridization of target & probe nucleic acid
    • 4. Detection of hybridization
  • 13.
    • I.PRODUCTION OF SINGLE STRAND PROBE NUCLEIC ACID
    • Depends on the sequence of intended target nucleic acid (ie. intended to use)
    • Probes are commercially synthesized and labeled with reporter molecule.
    • The user need to supply the manufacturer with the desired nucleotide base sequence
  • 14.
    • II. Preparation of target nucleic acid
    • Target nucleic acid must be single stranded & its base sequence integrity should be intact
    • Target preparation steps
    • 1. Enzymatic/chemical destruction of the
    • microbial envelope to release the target nucleic acid
    • 2. Stabilization of target nucleic acid to preserve structural integrity
    • 3. If target is DNA, denaturation into single strand ( generally heated to 94 C)
  • 15.
    • III. MIXTURE & HYBRIDIZATION OF
    • TARGET & PROBE
    • Environment in which probe & target are brought together is important
    • Hybridization stringency is most affected by
    • A. Salt concentrations in hybridization buffer
    • B. Temperature
    • C. Concentration of destabilizing agents
  • 16. NUCLEIC ACID PROBE & HYBRIDIZATION
  • 17.
    • IV. DETECTION OF HYBRIDIZATION
    • Depends on the reporter molecule used for labelling probe nucleic acid
    • 3 main reporter system used are
    • 1. Radioactive reporter
    • 2. Biotin-avidin reporter
    • 3. Chemiluminiscent reporter
  • 18.  
  • 19.  
  • 20.  
  • 21.  
  • 22. HYBRID CAPTURE TECHNIQUE:
    • Solution format
    • Solid support format
    • Southern hybridizations
    • Northern hybridizations
    • Dot Blot hybridization
    • In situ hybridizations
  • 23.
    • SOLUTION HYBRIDIZATION FORMAT
  • 24.
    • SOLID SUPPORT HYBRIDIZATION FORMAT
    • 1.Southern blot hybridization
    • 2. Northern blot hybridizations
    • 3.Dot Blot hybridization
    • 4.Insitu hybridization
  • 25. SOUTHERN BLOT HYBRIDIZATION
    • DNA fragments are separated by gel electrophoresis ,
    • usually an agarose gel.
    • Restriction fragments on the gel appears as a smear
    • rather than discrete bands.
    • Denature the fragments by incubation with NaOH.
    • Transferred to a membrane which is a sheet of
    • special blotting paper.
    • The DNA fragments will retain the same pattern
    • of separation they had on the gel.
  • 26.
    • The blot is incubated with many copies of labeled
    • probe which are single-stranded.
    • This probe will form base pairs with its complementary
    • DNA sequence to form a double-stranded DNA molecule.
    • The location of the probe is revealed by incubating it
    • with a colorless substrate that the attached enzyme
    • converts to a colored product
    • If the probe was labeled with radioactivity ,
    • it can expose X-ray film directly .
  • 27.  
  • 28.
    • SOUTHERN HYBRIDIZATION
  • 29. Southern blot/ DNA blot
  • 30. NORTHERN BLOT HYBRIDIZATION
  • 31. NORTHERN BLOT HYBRIDIZATION
  • 32. DOT BLOT HYBRIDIZATION
    • Detection of a given sequence of DNA/ RNA , not fractionated (not subjected to gel electrophoresis).
    • Method- DNA (or RNA) from different samples are transferred onto a nitrocellulose filter in form of dot .
    • The DNA is first denatured and then the filter is backed at 80ºC to fix the DNA firmly onto the filter.
    • Appropriate radioactive single stranded DNA probe is added
    • Hybridization with radioactive probes is detected by autoradiography .
    • the intensity of dot in the autoradiograph corresponds the extent to which DNA or RNA is represented in the sample.
  • 33.
    • INSITU HYBRIDIZATION
    • Allows a pathogen to be identified within the context of the pathologic lesion being
    • produced
    • Uses patient cells or tissues as the solid support phase
    • Combines the power of molecular diagnosis with the additional information that histopathology examination can provide.
  • 34. INSITU HYBRIDIZATION
    • Molecular pathology
    • FISH (fluorescent labeled probe)
    • CISH (chemiluminiscent probe)
  • 35. 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.
  • 36.  
  • 37.  
  • 38. CHEMILUMINISCENCE INSITU HYBRIDIZATION
    • Is performed using probes that are detected using enzymes with their appropriate chemiluminescent substrates.
    • The luminescent signal from the hybrid formation is detected, analyzed and measured with a high performance low light level imaging apparatus connected to an optical microscope and to a personal computer for quantitative image analysis.
    • This provides the estimation and quantification of nucleic acids present in tissue samples or cellular smears
  • 39. STEPS OF CISH
    • 1)  Identify Probe- – Prepare short sequences of DNA which are complementary to the target DNA sequences.
    • 2)  Label Probe- The probe is labeled with chemical hapten like digoxigenin.
    • 3)   Denature Chromosome and Probe - First, denature the chromosomes by way of heat or pH.  Then denature the probe via 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) Addition of chemiluminescent- Anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase are added to the slide.
    • 6)  Analysis – After washing the unhybridized probes, luminescent signal from the hybrid formation was detected, analyzed, and measured with luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis.
  • 40. Figure - Chemiluminescence in situ hybridization revealing increasing concentrations target DNA in a sample.
  • 41. DNA MICROARRAY
    • The core principle behind microarrays is hybridization between two DNA strands. The property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs .
    • A high number of complementary base pairs in a nucleotide sequence means tighter non- covalent bonding between the two strands.
    • Fluorescently labeled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases.
  • 42. DNA MICROARRAY
    • DNA oligonucleotide probes are attached to the solid support .(glass,nylon,silocon,ploypropylene etc).
    • Then unknown DNA undergo PCR to produce more number of target DNA fragments.
    • Labeled the target DNA with fluorescent dye .
    • Add this labeled DNA on to the known probe DNA.
    • Allow this hybridization to complete.
    • The hybridized probe can be detected in sensitive detection system.
  • 43. DNA MICROARRAY
  • 44. DNA MICROARRAY
  • 45. Fixed probes labeled target(sample) Different Features (eg bind different genes) Fully complementary strands bind strongly
  • 46.  
  • 47. AMPLIFICATION ASSAY
    • . PCR
    • LCR
    • NASBA
    • SDA
    • RAPD
    • RFLP
    • GENE SEQUENCING
  • 48.
    • WHY AMPLIFICATION??
    • If sufficient target nucleic acid is not present in the reaction, hybridization can give false negative results.
    • To circumvent this, nucleic acid amplification is used
    • How to amplify the nucleic acid?
    • By allowing repeated replication of target nucleic acid by the specific primer.
  • 49. POLYMERASE CHAIN REACTION(PCR)
    • Combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication that are applied repeatedly through numerous cycles for generation thousands to millions copies of a particular DNA sequence.
    • The method relies on thermal cycling , consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.
  • 50. PCR REQUIREMENT
    • DNA template that contains the DNA region (target) to be amplified.
    • primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target.
    • Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
    • Deoxynucleotide the building blocks from which the DNA polymerases synthesizes a new DNA strand.
    • Buffer solution , providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
    • Divalent cations , magnesium or manganese ions
    • Monovalent cation potassium ions.
  • 51.
    • THERMAL CYCLER
    • To maintain continuous reaction cycles, programmable thermal cyclers are used
  • 52. POLYMERASE CHAIN REACTION(PCR)
    • Basic steps:
    • Denaturation of the target (dsDNA)
    • Annealing of primers
    • Extension of primer-target Duplex
    • Detection of PCR products
  • 53.
    • DENATURATION OF NUCLEIC ACID
    • Target nucleic acid is added to the reaction mix that contains all necessary components of PCR to occur (primers, covalent ions, buffers, enzymes etc)
    • Denaturation of dsDNA to a single strand is accomplished by heating to 94 C
  • 54.
    • PRIMER ANNEALING
    • Primers are short single sequences of nucleotides (18-24 nucleotides)Selected specifically to flank the target sequence of interest.
    • When the primer pair is mixed with the denatured target DNA, one primer anneals to a specific site at 3’ end of the target strand, while the other primer anneals to a specific site at the 3’ end of the other , complementary target strand
    • Once the duplexes are formed (40-60c), the last step in cycle which mimics the DNA replication process, begins
  • 55. DENATURATION & PRIMER ANNEALING
  • 56.
    • EXTENSION OF PRIMER
    • TAQ POLYMERASE is the enzyme commonly used for primer extension, which occurs at 72 C
    • It can function efficiently at elevated temperature & withstand the denaturing temperature of 94 C through several cycles.
    • Annealing of primers to target sequence provides the necessary template format that allows DNA polymerase to add nucleotides to 3’ end of each primer & produce by extension a sequence complementary to target sequence
  • 57.
    • For each target sequence originally present in the PCR mixture, 2 double stranded fragments containing the target sequence are produced after one cycle
  • 58.
    • At the beginning of the second cycle of PCR, denaturation then produces 4 templates to which the primers will anneal.
    • Following extension at the end of the second cycle, there will be 4 double stranded fragments containing target nucleic acid
    • After 30 to 40 cycles, 10 7 to 10 8 target copies will be present in the reaction mixture
  • 59. POLYMERASE CHAIN REACTION(PCR) Sequential rounds
  • 60.
    • DETECTION OF PCR PRODUCT
    • (Post amplification analysis)
    • - Gel electrophoresis is the most common method
    • -Any of the basic methods previously described for detecting hybridization can also be adapted
  • 61.
    • GEL ELECTROPHORESIS
    • A portion of PCR mixture after amplification is subjected to gel electrophoresis
    • After electrophoresis, the gel is stained with ethidium bromide to visualize the amplicon
    • Using molecular weight size markers , the presence of amplicons of appropriate size is confirmed.
  • 62.
    • GEL ELECTROPHORESIS
  • 63. GEL ELECTROPHORESIS
  • 64. POLYMERASE CHAIN REACTION
    • Types of PCR
    • Nested PCR
    • Broad range PCR
    • Multiplex PCR
    • RT - PCR
    • Real Time PCR
  • 65. NESTED PCR
    • Two primers added sequentially
    • First amplicon serves as a target for second amplification
  • 66. 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.
  • 67.  
  • 68. BROAD RANGE PCR
    • Use of broad range specificity primers
    • Advantage: Primers target a larger group of microorganisms
    • Disadvantage: detection of phylogenetically related organisms but not those in the group of interest
  • 69. MULTIPLEX PCR
    • Multiple primers
    • Each primer can target a different organism
    • Used to detect:
      • Viral agents causing encephalitis
      • (HSV, Enteroviruses, West nile viruses)
      • Bacterial agents causing meningitis
      • (N. meningitidis ,M. tuberculosis etc)
      • Enteric pathogens (Salmonella - campylobacter ,Shigella,E.coli
  • 70. REVERSE TRANSCRIPTASE PCR
    • Target - RNA
    • Reverse transcriptase makes a cDNA copy of target
    • DNA polymerase makes a dsDNA
    • Routine PCR technique is done
    • Useful for detecting RNA viruses
  • 71.  
  • 72.  
  • 73.  
  • 74.  
  • 75. REAL TIME PCR
    • Real-time polymerase chain reaction , also called quantitative real time polymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chain reaction, is a laboratory technique based on the PCR , which is used to amplify and simultaneously quantify a targeted DNA molecule.
    • The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time.
    • The products are detected and measured in the real-time PCR thermocycler ,
  • 76. METHODS OF REALTIME PCR
    • Two different methods are used.
    • Method -1: - Real-time PCR using double-stranded DNA dyes.
    • In this method in addition to the all the PCR mix dsDNA dyes such as SYBR Green is added.
    • This dye will bind to all dsDNA PCR products , including nonspecific PCR products.
    • Method -2:- Fluorescent reporter probe method
    • In this method in addition to the all the PCR mix Fluorescent probes are added.
    • During the annealing stage of the PCR both probe and primers anneal to the DNA target.
    • Polymerization of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe , its 5'-3-exonuclease degrades the probe , physically separating the fluorescent reporter resulting in an increase in fluorescence .
  • 77. Real Time PCR
  • 78. LIGASE CHAIN REACTION
    • The ligase chain reaction (LCR) is a method of DNA amplification . While the better-known PCR carries out the amplification by polymerizing nucleotides, LCR instead amplifies the nucleic acid used as the probe. For each of the two DNA strands, two partial probes are ligated to form the actual one ; thus, LCR uses two enzymes: a DNA polymerase and a DNA ligase . Each cycle results in a doubling of the target nucleic acid molecule.
    • LCR is a good diagnostic potential but major limitation is the expensive requirement of two pair of deoxyoligomers.
  • 79. STEPS OF LIGASE CHAIN REACTION
    • Denaturation of target DNA at 94c.
    • Annealing of two pairs of synthetic deoxyoligomeres at40-60c.
    • Members of each pair bind in such a way that they are immediately adjacent and completely cover the target sequence on both separated DNA strands.
    • The oligomeres of each pair is then joined by a thermostable DNA ligase.
    • This doubles the target DNA molecules and complete the first LCR cycle
    • The second cycle is initiated by denaturation .
  • 80. LIGASE CHAIN REACTION
  • 81. NUCLEIC ACID SEQUENCE BASED AMPLIFICATION (NASBA)
    • NASBA is an ingenious method based on retroviral replication for amplification of RNA (or DNA with modification to this method).
    • This is more complex than PCR.But its does not require thermal cycling .
    • It proceeds rapidly and isothermally at 37c.
  • 82. STEPS OF NASBA The viral RNA strands are represented as the sense strand present in the original samples. Primer P1 binds to the RNA and is elongated by reverse transcriptase (AMV-RT). The RNA strand of the yielded DNA : RNA hybrid is hydrolyzed by RNase H. After the binding of P1, primer P2 can also bind. Primer P2 is then elongated by AMV – RT, yielding a double-stranded DNA molecule. Primer P1 is designed in such a manner that when it forms a double- stranded DNA, it codes for a T7 RNA polymerse Promoter site. This helps in generating antisense RNA copies using a DNA template. The new copies of DNA are generated using RNA. The process is same as followed for sense strand. Here in this case, P2 will bind first.
  • 83. NASBA
  • 84. STRAND DISPLACEMENT AMPLIFICATION(SDA)
    • This process consists of two phase-Target generation and second is amplification .
    • This process requires two primer pairs (B1,S1 and B2,S2) and two enzymes pairs( DNA polymerase and a restriction enzyme) , dATP and dAMP .
    • This process is efficient for small targets of less than 200 bp.
  • 85. STEPS OF SDA
    • Denaturation of the DNA and attachment of the primers.
    • Elongation of B1 and B2 primer to produce the target DNA.
    • Primer S1 and S2(having nick able site) binds with the target DNA and elongation takes place.
    • Incorporation of dAMP into the target DNA at the nick able site .
    • Nicking of the DNA strand(produced from primer s1 and s2) takes place and that DNA strands are separate out.
    • DNA polymerase again act at the nick able site and extends that strand again and the cycle continues.
  • 86.  
  • 87. RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)
    • It is a type of PCR reaction, but the segments of DNA that are amplified are random.
    • The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify.
    • By resolving the resulting patterns, a semi-unique profile can be gleaned in gel electrophoresis .
    • No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where.
  • 88. Molecular characterization of isolates using RAPD fingerprinting
    • RAPD fingerprints of reference strains
    • RAPD fingerprints of local isolates.    Patterns matching with that of reference strains indicated
  • 89. RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP )
    • Restriction enzymes are used to cut DNA into fragments (called restriction fragment length polymorphism) which may be characteristic at strain level.
    • These fragments are then separated by agarose gel electrophoresis , forming characteristic banding patterns .
    • Identification of the organism can then be established by comparing the pattern of unknown organism with that of known(reference) organism
  • 90. RFLP : Restriction Fragment Length Polymorphism
  • 91. sample
  • 92. GENE SEQUENCING
    • Sequence of nucleotide of the target gene is used for identification of organism.
    • Dideoxy nucleotides are added in the PCR mixture in addition to the fluorescent labeled nucleotides .
    • This Dideoxy nucleotide when added to the DNA process of elongation stops and denaturation takes place.
    • So in this way many copy of DNA fragments of DNA produced.
    • The sequence of nucleotides can be known by using automated DNA sequencer machine which uses capillary electrophoresis and laser printer .
  • 93. Gene sequence
  • 94. CONCLUSION
    • The molecular methods are a promising alternative that can substitute or complement the current reference method used in disease diagnosis.
    • More suitable and reliable results can be achieved in terms of speed and precision and can deter and enumerate specifically viable organism.
    • Sample preparation problem, contamination, entrenched attitude slow but cannot stop adoption of the molecular method of diagnosis.
  • 95. The man who removes a mountain begins by carrying away small stones