2Watson and CrickThe structure of DNA was described by British Scientists Watson and Crick as long double helix shaped with its sugar phosphate backbone on the outside and its bases on inside; the two strand of helix run in opposite direction and are anti-parallel to each other. The DNA double helix is stabilized by hydrogen bonds between the bases.
4DNAA purine always links with a pyrimidine base to maintain the structure of DNA.Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them.Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.
5Nucleic acid probeNucleic acid fragment, labelled by a radioisotope, biotin, etc., that is complementary to a sequence in another nucleic acid (fragment) and that will, by hydrogen binding to the latter, locate or identify it and be detected; a diagnostic technique based on the fact that every species of microbe possesses some unique nucleic acid sequences which differentiate it from all others, and can be used as identifying markers or "fingerprints."
6Nucleic acid probeNucleic acid fragment, labelled by a radioisotope, biotin, etc., that is complementary to a sequence in another nucleic acid (fragment) and that will, by hydrogen binding to the latter, locate or identify it and be detected; a diagnostic technique based on the fact that every species of microbe possesses some unique nucleic acid sequences which differentiate it from all others, and can be used as identifying markers or "fingerprints."
7Hybridization probeHybridization probe is a fragment of DNA or RNA of variable length (usually bases long), which is used to detect in DNA or RNA samples the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pairing due to complementarily between the probe and target.Dr.T.V.Rao MD
8Nucleic Acid ProbesAccu probes from Gene probe, which contains a chemiluminescent label and target the rRNA of the microorganisms of interest.It reads events in vivo or during the multiplication of organism.
9DNA is Endless structure The rungs of the ladder can occur in any order (as long as the base-pair rule is followed)Those 4 bases have endless combinations just like the letters of the alphabet can combine to make different words.
10DNA Replication So we remained what we were ? DNA replication is semi-conservative. That means that when it makes a copy, one half of the old strand is always kept in the new strand. This helps reduce the number of copy errors.So we remained what we were ?
11So we remained what we were Because of Our Genetic Materials
12DNA to RNADNA remains in the nucleus, but in order for it to get its instructions translated into proteins, it must send its message to the ribosome's, where proteins are made. The chemical used to carry this message is Messenger RNA
14Nucleic Acid Hybridizations The hybridization of a radioactive probe to filter bound DNA or RNA is one of the most informative experiments that is performed in molecular genetics. Two basic types of hybridizations are possible.Southern hybridization - hybridization of a probe to filter bound DNA; the DNA is typically transferred to the filter from a gelNorthern hybridization - hybridization of a probe to filter bound RNA; the RNA is typically transferred to the filter from a gel
15Western blottingWestern blotting is an Immunoblotting technique which rely on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules.In Western blotting, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein.The SDS PAGE technique is a prerequisite for Western blotting .
17Restriction fragment length polymorphism Sir Alec Jeffreys developed restriction fragment length polymorphism (RFLP), which quickly became the standard technique for DNA testing throughout the 1980s. RFLP provided the world with the first form of genetic testing based on DNA, the body's genetic material
18Restriction Fragment Length Polymorphism (RFLP) Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA.
19RFLPThe resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis, and transferred to a membrane via the Southern blot procedure.
21SpoligotypingSpoligotyping, a new method for simultaneous detection and typing of M. tuberculosis complex bacteria, has been recently developed. This method is based on polymerase chain reaction (PCR) amplification of a highly polymorphic direct repeat locus in the M. tuberculosis genome.
22Spoligotyping in Tuberculosis The well-conserved 36-bp direct repeats are interspersed with unique spacer sequences varying from 35 to 41 bp in size. Clinical isolates of MTC bacteria can be differentiated by the presence or absence of one or more spacers.
24DNA finger printingEvery one of our DNA is equal except for only about 0.10 %.DNA finger printing lies in uniqueness of those regions of DNA that do differ from person to person.Only 5 % of our DNA code rest do not code called in past as Junk DNA and contain repeated sequences of base pairsCalled as Variable number of tandem repeats contain 20 to 100 base pairs and the same sequence is repeated one to 3 times in a row
25Documentation of Finger printing for Records Finger print means translating all the variable number of tandem repeats to visible recordsAll VNTR is tested for restriction length polymorphism which differ from species to species.All the obtained material is blotted to Nylon or Nitrocellulose membrane ( Southern Blotting )
26RLFP to PCRIsolation of sufficient DNA for RFLP analysis is 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 in a shorter time.
28Molecular diagnostics – how it works Every organism contains some unique,species specific DNA sequencesMolecular diagnostics makes the species specific DNA visible
29PCR methods are rapid and sensitive PCR, as a specific, sensitive and rapid technique in the identification of the pathogen in the clinical specimen has been developed extensively over the past decade. Its value as a clinical tool is being increasingly recognized
30Polymerase Chain Reaction Methodology A Mile stone in Medical History He had the idea to use a pair of primers to bracket the desired DNA sequence and to copy it using DNA polymerase, a technique which would allow a small strand of DNA to be copied almost an infinite number of times.
31Dr. Kary Mullis, wins Nobel Prize in 1993 Kary received a Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR). The process, which Kary Mullis conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century.
33PCR Liberates a Innocent Prisoner KirkBloods worth caseA WatermanImprisoned for 9 years on wrong evidences of RapeUnmatched DNA by PCR makes a freeman
34Locates Genes for Color Blindness Color Blind British John Dalton died in 1844Request his eyes to be preservedAnd to be investigated why he confused scarlet with green, and pink with blueRecent PCR studies prove Dalton lacked a gene for making one of the three photo pigments essential for normal color vision.
35Color Blindness is x linked The genes for our red and green colour receptors are located on the X-chromosome, giving women a redundant set of receptor genes.This is why men are far more prone to colour-blindness than women.
36DNA – RNA - DNAIn Molecular biology, the polymerase chain reaction (PCR) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence.
37Common Tools of Molecular Biology Nucleic acid fractionationPolymerase chain reactionProbes, HybridizationVector, Molecular cloningNucleic acid enzymesMicroarrayDNA sequencingElectrophoretic separation of nucleic acidDetection of genes:*DNA: Southern blotting; inSitu hybridization; FISH Technique*RNA: Northern blotting*Protein: Western blotting, immunohistochemistry
38Current Uses of molecular Biology The most recent applied technologies, genetic engineering, DNA finger-printing in the social and forensic science, pre and postnatal diagnosis of inherited disease, gene therapy and drug Design.Molecular biology allows the laboratory to be predictive in nature, it gives information that the patients may be at risk for disease (future).Major tool in Diagnosis of Infectious
39Restriction 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. The similarity of the patterns generated can be used to differentiate species (and even strains) from one another.
40Restriction Endonulease Restriction endonucleases are enzymes that cleave DNA molecules at specific nucleotide sequences depending on the particular enzyme used. Enzyme recognition sites are usually 4 to 6 base pairs in length.The recognition sequences are randomly distributed through the DNA and recognizes different nucleotide sequences, and snips through DNA molecule.
41Taq polymeraseTaq polymerase is a thermos table DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in It is often abbreviated to "Taq Pol" (or simply "Taq"), and is frequently used in polymerase chain reaction (PCR), methods for greatly amplifying short segments of DNA
42Disadvantages of Taq Pol Taq mis-incorporates 1 base in 104.A 400 bp target will contain an error in 33% of molecules after 20 cycles.Error distribution will be random.
43Thermo cycler is Back bone of PCR methodology 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
46Cutting, pasting and amplifying is the basis of Reaction
47Denaturing Template Heat causes DNA strands to separate 5’3’3’5’Denature DNA strands 94oC3’5’5’3’
48Annealing Primers Primers bind to the template sequence Taq Polymerase recognizes double-stranded substrate3’5’3’5’Primers anneal 64oC5’3’3’5’3’5’5’3’
49Taq Polymerase Extends Taq Polymerase extends primerDNA is replicated3’5’5’3’3’5’5’3’Extend 72oC5’3’3’5’3’5’5’3’Repeat denaturing, annealing, and extending 30 cycles
50Molecular diagnostics is a set of methods to study primary structure (sequence) of DNAHybridization with complementary sequences-A-A-T-T-C-G-C-G-A-T-G-- T-T-A-A-G-C-G-C-T-A-C-Amplification (synthesis) of species specific sequencesPCR – polymerase chain reaction-A-A-T-T-C-G-C-G-A-T-G--A-A-T-T-C-G-C-G-A-T-G--A-A-T-T-C-G-C-G-A-T-G--A-A-T-T-C-G-C-G-A-T-G--A-A-T-T-C-G-C-G-A-T-G-The 7th Baltic Congress in Laboratory Medicine, Pärnu
55Candida infections can be specifically identified The fragments of 125-bp (EO3) and 317 bp (HSP) specific for C. albicans were used for amplification.
56Molecular methods proving highly Sensitive It has been postulated that DNA sequencing of the universal nested PCR product may allow identification of the causative organisms in a number of culture are few
57PCR helps in several critical Conditions PCR has also been evaluated in the diagnosis of fungal endophthalmitis using broad range primers as well as primers specific for C. albicans. Detection of fungal DNA by PCR in intraocular specimens will prove as a useful means of diagnosing endophthalmitis. It will greatly facilitate management decisions when conventional culture is negative.
58Advantages Molecular methods In-house (home-brew) PCR methods High sensitivity and specificityDetects pathogen, not immune responseQuick resultsHigh transport tolerationIn-house (home-brew) PCR methodsCost effectiveHigh sensitivityHigh qualityFast implementation of scientific discoveriesCustomer friendlyThe 7th Baltic Congress in Laboratory Medicine, Pärnu
60QIAGEN One Step RT-PCR Kit The QIAGEN One Step RT-PCR Kit is designed for easy and sensitive one-step RT-PCR using any RNA template. A unique enzyme combination and specially developed reaction buffer ensure efficient reverse transcription and PCR in one tube.
61RT-PCR in one step The Robus™ T I Kit is base RobusT RT-PCR Kits perform cDNA synthesis and PCR amplification of cDNA successively in a single tube during a continuous thermal cycling
62Nested polymerase chain reaction Nested polymerase chain reaction is a modification of polymerase chain reaction intended to reduce the contamination in products due to the amplification of unexpected primer binding sites.Nested polymerase chain reaction involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run products
63Loop Mediated Isothermal Amplification (LAMP) Loop mediated isothermal amplification is a simple, rapid, specific and cost effective nucleic acid amplification method characterized by use of 8 distinct regions on the target gene.The amplification proceeds at a constant temperature using strand displacement reaction.
64Multiplex PCRTaqMan probes and Molecular beacons allow multiple DNA species to be measured in the same sample ( Multiplex PCR) since fluorescent dyes with different emission spectra may be attached to different probes
65Multiplex PCR in Real Time Multiplex real time quantitative RT-PCR assays have been developed for simultaneous detection identification and quantification of HBV, HCV and HIV!In plasma and Serum samples.
66Prevention of Contamination in PCR Laboratory PCR contamination be considered as a form of infection. If standard sterile techniques that would be applied to tissue culture or microbiological manipulations are applied to PCR, then the risk of contamination will be greatly reduced. Above all else, common sense should prevail.
67Polymerase Chain Reaction Available for several infections …. Chlamydia trachomatisSlow growing Mycobacterium tuberculosisviruses like Herpes simplex virus ,Varicella Zoster virus .Adeno virus in our laboratory for corneal specimens
68Uses and Advantages in Testing by PCR Methods Clinical diagnostics: detection and quantification of infectious microorganisms, cancer cells and genetic disordersCapable of amplifying long targets, up to 6.0 kbOne-tube system allows rapid, sensitive and reproducible analysis of RNA with minimal risk of sample contaminationAmplifies products from a wide variety of total RNA or mRNA sources
69Disadvantages of PCR Methods Expensive to the Developing worldNeed well trained, ManpowerCoordination for quality controlAdoption to changing needsTimely technical supportFalse positive results due to Amplifications
70Key End User product feature requirements: Over 1400 molecular pathology labs in the US, with bulk of genetic testing being provided by a few big labs.Large Labs:Quest DiagnosticsLabCorpGenzymeMedium Labs:MayoARUP (Associated Regional and University Laboratories Pathologist)MD AndersonRegional clinics and hospital LaboratoriesKey End User product feature requirements:Clinical Practicality of testStrong client portfolio and relationship with laboratoriesResult turnaround time
71Important issue in genetic testing market is deciding which testing platform or technology will have highest adoption rates in Clinical labsTechnology issues are throughput, sample concentration, procedure sensitivity and specificityEconomic issues are costs of instrument and reagents, current platforms available in the lab, and ease of use of the proceduresGenetic testing looks for genetic marker such as Single Nucleotide Polymorphism (SNP) , Restriction Fragment length Polymorphism (RFLP) or short tandem repeat (STR)Theoretically any marker can be identified through any of the platformsReal- Time Polymerase Chain Reaction ( RTPC)Capillary ElectrophoresisMicro-arrayMultiplexing Instruments
72Restriction Fragment length Polymorphism (RFLP), or Important issue in genetic testing market is deciding which testing platform or technology will have highest rates in Clinical labsTechnology issues are throughput, sample concentration, procedure sensitivity and specificityEconomic issues are costs of instrument and reagents, current platforms available in the lab, and ease of use of the proceduresGenetic testing looks for genetic marker such as Single Nucleotide Polymorphism (SNP)Restriction Fragment length Polymorphism (RFLP), orShort tandem repeat (STR)Theoretically any marker can be identified through any of the platformsReal- Time Polymerase Chain Reaction ( RTPC)Capillary ElectrophoresisMicro-arrayMultiplexing Instruments
73Electrophoresis Platform: A method of detection for specific gene sequences using gel electrophoresisCurrently high availability of electrophoresis instrumentation in labs means that kits designed for this platform should have the most rapid uptake.Least expensive- $15K US for state of the art equipmentElectrophoresis also offers low costs per tests - kit cost $80/patientVersatile in number of different applicationMajor disadvantage is labor intensity
74RT-PCR Platform:A method for rapid and simultaneous amplification, detection and quantification of gene fragments or gene expressionSlower adoption than electrophoresis due to the cost of machinery - $30KExpected to have significant uptake because of prevalence in SNP testingCurrently many specialty labs like Quest Diagnostics have high RT-PCR usageCost expected to drop
75Microarray Platform:A method for rapid detection of multiple simultaneous gene fragments or gene expression. Probes that react to patient’s genetic material are arranged in grid pattern on glass or plastic platform. Resulting matches constitute a positive test, which are readily identifiable.Most Expensive $150K-$180K + $500 per testReaction indicate particular genetic sequences, such as those related to diseases, or how people will respond to certain medications. Microarrays also can enable researchers to see which genes are being switched on and off under different medical conditions.Currently does not have many clinical applications, so drawback for laboratories to invest in platformRoche’s AmpliChip is only FDA approved microarray test for diagnostic use
76Multiplexing Platform: A method for simultaneous detection of specific gene fragments, gene expression, immune response proteins and enzymes. Multifunction beads are manipulated to bind and signal the specific presence of a a variety of substratesMore versatile than microarrayAllows more variation with regards to type of test - up to 100 assays to be preformed simultaneously on one sample.Slower than competing technologies howeverMultiplexing technology are expected to gain field.FDA awarded its first approval for a CF diagnostic test to TM Bioscience’s multiplexing platform based test.
77Regulatory IssuesFDA regulates reagents, kits and instruments sold to and performed by clinical labs.Diagnostic tests can be sold as Analyte Specific Reagents (ASR) to highly sophisticated CLIA-certified labs. FDA exercised restrain in regulating home- brew tests performed by these labs. No specific clinical, validity or performance claims are allowed for unregulated ASRs. Potential liabilities if a lab chooses to provide an ASR rather than a 510(k)-cleared alternative.Receiving a 510(k) FDA approval is a significant market barrier:Four FDA approved genetic diagnostic tests:Roche AmpliChip, used to individualize dosage of antidepressants, antipsychotics, beta-blockers, and some chemotherapy drugsTM Bioscience (Toronto, Canada) TAG-IT assay for detecting cystic fibrosisVisual Genetics (Toronto, Canada acquired by Bayer in ‘02) TRUGENE HIV-1 Genotyping Kit, used to detect variations in the genome of the human immunodeficiency virus that make the virus resistant to some anti-retroviral drugs.Third Wave’s Invader assay detects variations in a gene that produces the enzyme UDP-glucuronosyltransferase, and predicts adverse events.