Biology to Molecular Biology Emerging Trands

8/3/2019 Biology to Molecular Biology Emerging Trands

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Biology to Molecular BiologyEmerging Trends in Diagnosis

Dr. T.V.Rao MD

Dr.T.V.Rao MD 1


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From Biology to Molecular Biology

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Watson and Crick

The structure of DNA was described byBritish Scientists Watson and Crickas longdouble helix shaped with its sugar

phosphate backbone on the outside andits bases on inside; the two strand ofhelix run in opposite direction and are

anti-parallel to each other. The DNAdouble helix is stabilized by hydrogenbonds between the bases

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Watson and Crick discovers DNA

Feb 28th 1953

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DNA

A purine always links with apyrimidine base tomaintain the structure ofDNA.

Adenine ( A ) binds to Thymine( T ), with two hydrogenbonds between them.

Guanine ( G ) binds toCytosine ( C ), with threehydrogen bonds betweenthem.

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Important Methods

1 Nucleic acidprobes

2 Hybridcapture

3 Branched

chain DNA4 Situ

hybridization

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Nucleic acid probe

Nucleic acid fragment, labelled by aradioisotope, biotin, etc., that is complementaryto a sequence in another nucleic acid (fragment)

and that will, by hydrogen binding to the latter,locate or identify it and be detected; adiagnostic technique based on the fact thatevery species of microbe possesses some

unique nucleic acid sequences whichdifferentiate it from all others, and can be usedas identifying markers or "fingerprints."

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Hybridization probe

Hybridization probe is a fragment of DNA orRNA of variable length (usually 100-1000 baseslong), which is used to detect in DNA or RNA

samples the presence of nucleotide sequences(the DNA target) that are complementary to thesequence in the probe. The probe therebyhybridizes to single-stranded nucleic acid (DNA

or RNA) whose base sequence allows probe-target base pairing due to complementarilybetween the probe and target.

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Nucleic Acid Probes

Accu probes from Gene

probe, In which itcontainChemiluminescent label

and target the rRNA ofthe Microorganisms ofinterest.

It reads events in vivo

or during themultiplication oforganism.

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DNA is Endless structure

The rungs of the laddercan occur in any order(as long as the base-

pair rule is followed) Those 4 bases have

endless combinationsjust like the letters of

the alphabet cancombine to makedifferent words.

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DNA Replication

DNA replication issemi-conservative.That means that whenit makes a copy, one

half of the old strand isalways kept in the newstrand. This helpsreduce the number of

copy errors. So we remained

what we were ?

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So we remained what we were

Because of Our Genetic Materials

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DNA to RNA

DNA remains in thenucleus, but in order forit to get its instructionstranslated into proteins,it must send itsmessage to theribosome's, whereproteins are made. Thechemical used to carrythis message isMessenger RNA

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Blotting technique

Southern Blot

It is used to detect DNA.

Northern Blot

It is used to detect RNA.

Western blot

It is used to detect protein.

TYPES OF BLOTTING TECHNIQUES

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Nucleic Acid Hybridizations

The hybridization of a radioactive probe tofilter bound DNA or RNA is one of the mostinformative experiments that is performed inmolecular genetics. Two basic types ofhybridizations are possible.

Southern hybridization - hybridization of aprobe to filter bound DNA; the DNA is typicallytransferred to the filter from a gel

Northern hybridization - hybridization of aprobe to filter bound RNA; the RNA is typicallytransferred to the filter from a gel

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Western blotting

Western blotting is an Immunoblotting techniquewhich rely on the specificity of binding between amolecule of interest and a probe to allow

detection of the molecule of interest in a mixtureof many other similar molecules.

In Western blotting, the molecule of interest isa protein and the probe is typically an antibody

raised against that particular protein. The SDS PAGE technique is a prerequisite for

Western blotting .

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Western Blotting

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Restriction fragment length

polymorphism

Sir Alec Jeffreys developed restrictionfragment length polymorphism (RFLP), whichquickly became the standard technique for

DNA testing throughout the 1980s. RFLPprovided the world with the first form ofgenetic testing based on DNA, the body'sgenetic material

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Restriction Fragment Length

Polymorphism (RFLP)

Restriction FragmentLength Polymorphism(RFLP) is a techniquein which organismsmay be differentiatedby analysis of patternsderived from cleavageof their DNA.

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RFLP

The resulting DNAfragments are thenseparated by lengththrough a processknown as agarose gelelectrophoresis, andtransferred to amembrane via theSouthern blotprocedure.

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Spoligotyping Spoligotyping, a new method for

simultaneous detection and typing of

M. tuberculosiscomplex bacteria, hasbeen recently developed. This

method is based on polymerase chain

reaction (PCR) amplification of ahighly polymorphic direct repeat locus

in the M. tuberculosisgenome.

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Spoligotyping in Tuberculosis

The well-conserved 36-bp direct repeats areinterspersed withunique spacersequences varying from35 to 41 bp in size.Clinical isolates of MTCbacteria can bedifferentiated by thepresence or absence ofone or more spacers.

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Results analyzed by Computer

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DNA finger printing

Every one of our DNA is equal except for only about0.10 %.

DNA finger printing lies in uniqueness of thoseregions of DNA that do differ from person to person.

Only 5 % of our DNA code rest do not code called inpast as Junk DNA and contain repeated sequencesof base pairs

Called as Variable number of tandemrepeats contain 20 to 100 base pairs and thesame sequence is repeated one to 3 times in a row

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Documentation of Finger printing

for Records Finger print means translating all the variable

number of tandem repeats to visible records

All VNTR is tested for restriction length

polymorphism which differ from species tospecies.

All the obtained material is blotted to Nylon or

Nitrocellulose membrane ( Southern Blotting )

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RLFP to PCR

Isolation of sufficient DNA for RFLP analysisis time-consuming and labour intensive.However, PCR can be used to amplify very

small amounts of DNA, usually in 2-3 hours,to the levels required for RFLP analysis.Therefore, more samples can be analysed ina shorter time.

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Opportunistic Infections are

Difficult to Diagnose Opportunistic pathogenic agents are

increasingly encountered in ocularinfections due to widespread use of topical

and systemic immunosuppressive agents,increasing numbers of patients with (HIV)infection and with organ transplants whoare on immunosuppressive therapy and

cause ocular infections due to increaseduse of contact lens.

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Cataract Surgery Legal Implications

The dreaded

infectionsendophthalmitis

following cataractextraction and lensimplantation often

are caused byopportunisticpathogens

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Diagnostic methods in

microbiologyTask of the method to make the microorganismvisible and measureableDifficult on several Occasions

Microscopy

Cultivation

Bio-testing

Immunological methodsBiochemical methods

Molecular methods

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Culturing continues to be Less

sensitive Culture of intraocular

specimens is consideredas the gold standard inthe diagnosis of

endophthalmitis. underthe most appropriatecare, traditionalmicrobiological methods

yield positive results inonly 60-70% of theclinically typical cases ofendophthalmitis.

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Prior Antibiotic therapy reduces the

isolate rate Prior antibiotic therapy,

small number oforganisms in thesamples, possible

localized nature ofinfections in the lenscapsule and fastidiousgrowth requirement of the

offending organisms, theorganism not beingrecovered in roughlyaround 30-40% of thecases.

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Molecular diagnosticshow it works

Every organismcontains someunique,

species specific DNAsequences

Molecular diagnostics

makes the speciesspecific DNA visible

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PCR methods are rapid and sensitive

PCR, as a specific,sensitive and rapidtechnique in theidentification of thepathogen in the clinicalspecimen has beendeveloped extensivelyover the past decade.Its value as a clinicaltool is beingincreasingly recognized

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Polymerase Chain Reaction Methodology A

Mile stone in Medical History

He had the idea to usea pair of primers tobracket the desiredDNA sequence and tocopy it using DNApolymerase, atechnique which wouldallow a small strand ofDNA to be copiedalmost an infinitenumber of times.

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Dr. Kary Mullis, wins Nobel Prizein

1993

Kary received a NobelPrize in chemistry in1993, for his invention ofthe polymerase chain

reaction (PCR). Theprocess, which KaryMullis conceptualized in1983, is hailed as one of

the monumental scientifictechniques of thetwentieth century.

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PCR Liberates a Innocent

Prisoner KirkBloods worth

case

A Waterman

Imprisoned for 9years on wrongevidences of Rape

Unmatched DNAby PCR makes afreeman

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Locates Genes for Color Blindness

Color Blind British JohnDalton died in 1844

Request his eyes to bepreserved

And to be investigated whyhe confused scarlet withgreen, and pink with blue

Recent PCR studies proveDalton lacked a gene formaking one of the threephoto pigments essential fornormal color vision.

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Color Blindness is x linked

The genes for our redand green colourreceptors are locatedon the X-chromosome,giving women aredundant set ofreceptor genes.

This is why men are farmore prone to colour-blindness than women.

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DNARNA - DNA

In Molecular biology,the polymerase chainreaction (PCR) is atechnique to amplify asingle or few copies ofa piece of DNA acrossseveral orders ofmagnitude, generatingmillions or more copiesof a particular DNA

sequence. Doctortvraos e learning series

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Common Tools of Molecular Biology

Nucleic acid fractionation Polymerase chain reaction Probes, Hybridization Vector, Molecular cloning Nucleic acid enzymes Microarray DNA sequencing Electrophoretic separation of nucleic acid Detection of genes:

*DNA:Southern blotting; inSitu hybridization; FISH

Technique*RNA:Northern blotting*Protein:Western blotting, immunohistochemistry

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Current Uses of molecular

BiologyThe most recent applied technologies, genetic

engineering, DNA finger-printing in the socialand forensic science, pre and postnataldiagnosis of inherited disease, gene therapy and

drug Design.

Molecular biology allows the laboratory to bepredictive in nature, it gives information that the

patients may be at risk for disease (future).Major tool in Diagnosis of Infectious

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Restriction Endonulease

If two organisms differ in the distance

between sites of cleavage of a

particular restriction endonuclease,the length of the fragments produced

will differ when the DNA is digested

with a restriction enzyme. Thesimilarity of the patterns generated

can be used to differentiate species

(and even strains) from one another.Dr.T.V.Rao MD 45


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Restriction Endonulease

Restriction endonucleases are enzymes thatcleave DNA molecules at specific nucleotidesequences depending on the particularenzyme used. Enzyme recognition sites areusually 4 to 6 base pairs in length.

The recognition sequences are randomlydistributed through the DNA and recognizes

different nucleotide sequences, and snipsthrough DNA molecule.

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Taq polymerase

Taq polymerase is a thermos table DNApolymerase named after the thermophilicbacterium Thermus aquaticusfrom which

it was originally isolated by Thomas D.Brock in 1965. It is often abbreviated to"Taq Pol" (or simply "Taq"), and is

frequently used in polymerase chainreaction (PCR), methods for greatlyamplifying short segments of DNA

Doctortvraos e learning series

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Disadvantages of Taq Pol

Taqmis-incorporates 1base in 104.

A 400 bp target willcontain an error in 33%of molecules after 20cycles.

Error distribution will berandom.

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Thermo cycler is Back bone of PCR

methodology

The method relies

on thermal cycling,consisting of cycles

of repeated heatingand cooling of thereaction for DNA

melting andenzymaticreplication of the

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PCR - Three basic Steps

Cut

Paste

Amplify

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PCR Primers

TTAACGGCCTTAA . . . TTTAAACCGGTT

AATTGCCGGAATT . . . . . . . . . .>

and


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Cutting, pasting and amplifying is

the basis of Reaction

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D t i T l t


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Denaturing Template

Heat causes DNA strands to separate

3

5

5

3

Denature DNA strands 94oC

5

3

3

5

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Annealing Primers


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Annealing Primers

Primers bind to the template sequence

Taq Polymerase recognizes double-stranded substrate

3

5

5

3

Primers anneal 64oC

3

5

5

33 535

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Taq Polymerase Extends


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Taq Polymerase Extends

3

5

3 535

Extend 72oC

3

53 535

5

3

5

3

Taq Polymerase extends primer

DNA is replicated

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Molecular diagnostics is a set of methodsto study primary structure (sequence) of DNA

Hybridization with complementary sequences

Amplification (synthesis) of species specific sequencesPCR polymerase chain reaction

The 7th Baltic Congress in Laboratory Medicine, Prnu 11.09.2004

-A-A-T-T-C-G-C-G-A-T-G-- T-T-A-A-G-C-G-C-T-A-C-

-A-A-T-T-C-G-C-G-A-T-G-





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PCR i l d M l i li


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PCR occurs in cycles and Multiplies

the DNA

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Dr.T.V.Rao MD 58

A li i f PCR


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Applications of PCR

The standard

specimen

procedure canquantitate HIV-

1 RNA in a

range of 400-75,000

copies/mL.Dr.T.V.Rao MD 59

Ad f PCR


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Advantages of PCR

Speed

Ease of use

Sensitivity

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C did i f i b ifi ll


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Candida infections can be specifically

identified

The fragments

of 125-bp

(EO3) and 317bp (HSP)

specific for C.

albicans wereused for

amplification.Dr.T.V.Rao MD 61

M l l h d i hi hl


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Molecular methods proving highly

Sensitive

It has been postulatedthat DNA sequencingof the universal

nested PCR productmay allowidentification of thecausative organisms

in a number of cultureare few

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PCR h l i l i i l


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PCR helps in several critical

Conditions PCR has also been

evaluated in the diagnosisof fungal endophthalmitisusing broad range primersas well as primers specific

for C. albicans. Detection offungal DNA by PCR inintraocular specimens willprove as a useful means ofdiagnosing endophthalmitis.

It will greatly facilitatemanagement decisionswhen conventional cultureis negative.

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Ad t


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The 7th Baltic Congress in Laboratory Medicine, Prnu 11.09.2004

Advantages

Molecular methodsHigh sensitivity and specificityDetects pathogen, not immune response

Quick results

High transport toleration

In-house (home-brew) PCR methodsCost effective

High sensitivity

High qualityFast implementation of scientific discoveries

Customer friendly

R&D is absolutely necessary

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Dr.T.V.Rao MD 65

QIAGEN O St RT PCR Kit


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QIAGEN One Step RT-PCR Kit

The QIAGEN One StepRT-PCR Kit is designedfor easy and sensitiveone-step RT-PCR using

any RNA template. Aunique enzymecombination andspecially developedreaction buffer ensure

efficient reversetranscription and PCRin one tube.

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RT PCR in n t p


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RT-PCR in one step

The Robus T I Kit is base

RobusT RT-PCRKits perform cDNAsynthesis and PCR

amplification ofcDNA successivelyin a single tubeduring a continuous

thermal cycling

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N t d l h i ti


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Nested polymerase chain reaction

Nested polymerase chain reaction is amodification of polymerase chain reaction

intended to reduce the contamination in

products due to the amplification of unexpectedprimer binding sites.

Nested polymerase chain reaction involves twosets of primers, used in two successive runs of

polymerase chain reaction, the second setintended to amplify a secondary target within thefirst run products

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Loop Mediated Isothermal


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Loop Mediated Isothermal

Amplification (LAMP)

Loop mediated isothermal

amplification is a simple, rapid,

specific and cost effective nucleicacid amplification method

characterized by use of 8 distinct

regions on the target gene.

The amplification proceeds at a

constant temperature using strand

displacement reaction.Dr.T.V.Rao MD 69

M l i l PCR


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Multiplex PCR

TaqMan probes andMolecular beaconsallow multiple DNAspecies to be

measured in thesame sample (Multiplex PCR) sincefluorescent dyes with

different emissionspectra may beattached to differentprobes

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M ltiple PCR in Re l Time


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Multiplex PCR in Real Time

Multiplex real timequantitative RT-PCRassays have been

developed forsimultaneousdetectionidentification and

quantification of HBV,HCV and HIV-! Inplasma and Serumsamples.

Doctortvraos e learning series Dr.T.V.Rao MD 71

Prevention of Contamination in PCR


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Prevention of Contamination in PCR

Laboratory

PCR contamination be considered as

a form ofinfection. If standard steriletechniques that would be applied totissue culture or microbiological

manipulations are applied to PCR,

then the risk of contamination will begreatly reduced. Above all else,

common sense should prevail.

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Polymerase Chain Reaction


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Polymerase Chain Reaction

Available for several infections.

Chlamydia trachomatis

Slow growing Mycobacterium

tuberculosis viruses like Herpes simplex virus ,

Varicella Zoster virus .

Adeno virus in our laboratory forcorneal specimens

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Uses and Advantages in Testing by


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Uses and Advantages in Testing by

PCR Methods

Clinical diagnostics: detection and quantificationof infectious microorganisms, cancer cells andgenetic disorders

Capable of amplifying long targets, up to 6.0 kb One-tube system allows rapid, sensitive and

reproducible analysis of RNA with minimal risk ofsample contamination

Amplifies products from a wide variety of totalRNA or mRNA sources

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Disadvantages of PCR Methods


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Disadvantages of PCR Methods

Expensive to the Developing world

Need well trained, Manpower

Coordination for quality control

Adoption to changing needs

Timely technical support

False positive results due to Amplifications

Above all dedicated Staff

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Molecular Epidemiology in Eye Care


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Molecular Epidemiology in Eye Care

In several world health funded projectsmolecular methods are used for

1 Plasmid analysis

2 Genomic fingerprinting3 Emerging drug resistance

4 Polymerase chain reaction to identifyemerging and remerging microbes,

5 Determination of antibiotic resistancepatterns.

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Be Familiar with Molecular Biology or


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Be Familiar with Molecular Biology or

???

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Bioinformatics may take over


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Bioinformatics may take over

Biology ???

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Programme created by Dr.T.V.Rao MDfor basic awareness on

Molecular Methods in Diagnosis Email

[email protected]

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Biology to Molecular Biology Emerging Trands