Biology to Molecular Biology, Emerging Diagnostic Trends

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Biology to Molecular Biology, Emerging Diagnostic Trends

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Biology to Molecular Biology Emerging Trends in Diagnosis Dr. T.V.Rao MD:

Biology to Molecular Biology Emerging Trends in Diagnosis Dr. T.V.Rao MD Dr.T.V.Rao MD 1

From Biology to Molecular Biology:

From Biology to Molecular Biology Dr.T.V.Rao MD 2

Watson and Crick:

Watson and Crick The 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 Dr.T.V.Rao MD 3

Watson and Crick discovers DNA Feb 28th 1953:

Watson and Crick discovers DNA Feb 28 th 1953 Dr.T.V.Rao MD 4

DNA:

DNA A 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. Dr.T.V.Rao MD 5

Important Methods:

Important Methods 1 Nucleic acid probes 2 Hybrid capture 3 Branched chain DNA 4 Situ hybridization Dr.T.V.Rao MD 6

Nucleic acid probe :

Nucleic acid probe Nucleic 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." Dr.T.V.Rao MD 7

Hybridization probe:

Hybridization probe H ybridization probe is a fragment of DNA or RNA of variable length (usually 100-1000 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 8

Nucleic Acid Probes:

Nucleic Acid Probes Accu probes from Gene probe, In which it contain Chemiluminescent label and target the rRNA of the Microorganisms of interest. It reads events in vivo or during the multiplication of organism. Dr.T.V.Rao MD 9

DNA is Endless structure:

DNA 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. Dr.T.V.Rao MD 10

DNA Replication:

DNA Replication 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 ? Dr.T.V.Rao MD 11

So we remained what we were Because of Our Genetic Materials:

So we remained what we were Because of Our Genetic Materials Dr.T.V.Rao MD 12

DNA to RNA :

DNA to RNA DNA 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 Dr.T.V.Rao MD 13

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TYPES OF BLOTTING TECHNIQUES Dr.T.V.Rao MD 14

Nucleic Acid Hybridizations :

Nucleic 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 gel Northern hybridization - hybridization of a probe to filter bound RNA; the RNA is typically transferred to the filter from a gel Dr.T.V.Rao MD 15

Western blotting:

Western blotting Western 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 . Dr.T.V.Rao MD 16

Western Blotting:

Western Blotting Dr.T.V.Rao MD 17

Restriction fragment length polymorphism :

Restriction 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 Dr.T.V.Rao MD 18

Restriction Fragment Length Polymorphism (RFLP) :

Restriction 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. Dr.T.V.Rao MD 19

RFLP:

RFLP The 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. Dr.T.V.Rao MD 20

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Dr.T.V.Rao MD 21

Spoligotyping:

Spoligotyping Spoligotyping, 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 . Dr.T.V.Rao MD 22

Spoligotyping in Tuberculosis:

Spoligotyping 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. Dr.T.V.Rao MD 23

Results analyzed by Computer:

Results analyzed by Computer Dr.T.V.Rao MD 24

DNA finger printing:

DNA finger printing Every 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 pairs Called 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 Dr.T.V.Rao MD 25

Documentation of Finger printing for Records:

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 to species. All the obtained material is blotted to Nylon or Nitrocellulose membrane ( Southern Blotting ) Dr.T.V.Rao MD 26

RLFP to PCR:

RLFP to PCR Isolation 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. Dr.T.V.Rao MD 27

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Dr.T.V.Rao MD 28

Opportunistic Infections are Difficult to Diagnose:

Opportunistic Infections are Difficult to Diagnose Opportunistic pathogenic agents are increasingly encountered in ocular infections due to widespread use of topical and systemic immunosuppressive agents, increasing numbers of patients with (HIV) infection and with organ transplants who are on immunosuppressive therapy and cause ocular infections due to increased use of contact lens. Dr.T.V.Rao MD 29

Cataract Surgery Legal Implications:

Cataract Surgery Legal Implications The dreaded infections endophthalmitis following cataract extraction and lens implantation often are caused by opportunistic pathogens Dr.T.V.Rao MD 30

Diagnostic methods in microbiology :

Diagnostic methods in microbiology Task of the method – to make the microorganism “visible” and “measureable” Difficult on several Occasions Microscopy Cultivation Bio-testing Immunological methods Biochemical methods Molecular methods Dr.T.V.Rao MD 31

Culturing continues to be Less sensitive:

Culturing continues to be Less sensitive Culture of intraocular specimens is considered as the gold standard in the diagnosis of endophthalmitis. under the most appropriate care, traditional microbiological methods yield positive results in only 60-70% of the clinically typical cases of endophthalmitis. Dr.T.V.Rao MD 32

Prior Antibiotic therapy reduces the isolate rate:

Prior Antibiotic therapy reduces the isolate rate Prior antibiotic therapy, small number of organisms in the samples, possible localized nature of infections in the lens capsule and fastidious growth requirement of the offending organisms, the organism not being recovered in roughly around 30-40% of the cases. Dr.T.V.Rao MD 33

Molecular diagnostics – how it works:

Molecular diagnostics – how it works Every organism contains some unique , species specific DNA sequences Molecular diagnostics makes the species specific DNA visibl e Dr.T.V.Rao MD 34

PCR methods are rapid and sensitive:

PCR 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 Dr.T.V.Rao MD 35

Polymerase Chain Reaction Methodology A Mile stone in Medical History:

Polymerase 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. Dr.T.V.Rao MD 36

Dr. Kary Mullis, wins Nobel Prize in 1993:

Dr. 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. Dr.T.V.Rao MD 37

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Dr.T.V.Rao MD 38

PCR Liberates a Innocent Prisoner:

PCR Liberates a Innocent Prisoner KirkBloods worth case A Waterman Imprisoned for 9 years on wrong evidences of Rape Unmatched DNA by PCR makes a freema n Dr.T.V.Rao MD 39

Locates Genes for Color Blindness:

Locates Genes for Color Blindness Color Blind British John Dalton died in 1844 Request his eyes to be preserved And to be investigated why he confused scarlet with green, and pink with blue Recent PCR studies prove Dalton lacked a gene for making one of the three photo pigments essential for normal color vision. Dr.T.V.Rao MD 40

Color Blindness is x linked:

Color 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. Dr.T.V.Rao MD 41

DNA – RNA - DNA:

DNA – RNA - DNA In 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. Doctortvrao’s ‘e’ learning series Dr.T.V.Rao MD 42

Common Tools of Molecular Biology:

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 Dr.T.V.Rao MD 43

Current Uses of molecular Biology:

Current 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 Dr.T.V.Rao MD 44

Restriction Endonulease:

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. The similarity of the patterns generated can be used to differentiate species (and even strains) from one another. Dr.T.V.Rao MD 45

Restriction Endonulease:

Restriction 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. Dr.T.V.Rao MD 46

Taq polymerase :

Taq polymerase Taq 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 1965. 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 Doctortvrao’s ‘e’ learning series Dr.T.V.Rao MD 47

Disadvantages of Taq Pol:

Disadvantages of Taq Pol Taq mis-incorporates 1 base in 10 4 . A 400 bp target will contain an error in 33% of molecules after 20 cycles. Error distribution will be random. Dr.T.V.Rao MD 48

Thermo cycler is Back bone of PCR methodology:

Thermo 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 Doctortvrao’s ‘e’ learning series Dr.T.V.Rao MD 49

PCR - Three basic Steps:

PCR - Three basic Steps Cut Paste Amplify Dr.T.V.Rao MD 50

PCR Primers:

PCR Primers TT AA C GG CC TT AA . . . TTT AAA CC GG TT AA TT G CC GG AA TT . . . . . . . . . .> and <. . . . . . . . . . AAA TTT GG CC AA TT AA C GG CC TT AA . . . TTT AAA CC GG TT Dr.T.V.Rao MD 51

Cutting, pasting and amplifying is the basis of Reaction:

Cutting, pasting and amplifying is the basis of Reaction Dr.T.V.Rao MD 52

Denaturing Template:

Denaturing Template Heat causes DNA strands to separate 3’ 5’ 5’ 3’ Denature DNA strands 94 o C 5’ 3’ 3’ 5’ Dr.T.V.Rao MD 53

Annealing Primers:

Annealing Primers Primers bind to the template sequence Taq Polymerase recognizes double-stranded substrate 3’ 5’ 5’ 3’ Primers anneal 64 o C 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ Dr.T.V.Rao MD 54

Taq Polymerase Extends:

Taq Polymerase Extends 3’ 5’ 3’ 5’ 3’ 5’ Extend 72 o C 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ Taq Polymerase extends primer DNA is replicated Repeat denaturing, annealing, and extending 30 cycles Dr.T.V.Rao MD 55

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Molecular diagnostics is a set of methods to study primary structure (sequence) of DNA Hybridization with complementary sequences Amplification (synthesis) of species specific sequences PCR – polymerase chain reaction The 7th Baltic Congress in Laboratory Medicine, Pärnu 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- -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- Dr.T.V.Rao MD 56

PCR occurs in cycles and Multiplies the DNA:

PCR occurs in cycles and Multiplies the DNA Dr.T.V.Rao MD 57

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Dr.T.V.Rao MD 58

Applications of PCR:

Applications of PCR The standard specimen procedure can quantitate HIV-1 RNA in a range of 400-75,000 copies/mL . Dr.T.V.Rao MD 59

Advantages of PCR:

Advantages of PCR Speed Ease of use Sensitivity Dr.T.V.Rao MD 60

Candida infections can be specifically identified:

Candida infections can be specifically identified The fragments of 125-bp (EO3) and 317 bp (HSP) specific for C. albicans were used for amplification. Dr.T.V.Rao MD 61

Molecular methods proving highly Sensitive:

Molecular 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 Dr.T.V.Rao MD 62

PCR helps in several critical Conditions:

PCR 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. Dr.T.V.Rao MD 63

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The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004 Advantages Molecular methods High sensitivity and specificity Detects pathogen, not immune response Quick results High transport toleration In-house (home-brew) PCR methods Cost effective High sensitivity High quality Fast implementation of scientific discoveries Customer friendly R&D is absolutely necessary Dr.T.V.Rao MD 64

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QIAGEN One Step RT-PCR Kit :

QIAGEN 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. Dr.T.V.Rao MD 66

RT-PCR in one step The Robus™ T I Kit is base :

RT-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 Dr.T.V.Rao MD 67

Nested polymerase chain reaction:

Nested 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 Dr.T.V.Rao MD 68

Loop Mediated Isothermal Amplification (LAMP):

Loop 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. Doctortvrao’s ‘e’ learning series Dr.T.V.Rao MD 69

Multiplex PCR:

Multiplex PCR TaqMan 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 Dr.T.V.Rao MD 70

Multiplex PCR in Real Time:

Multiplex 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. Doctortvrao’s ‘e’ learning series Dr.T.V.Rao MD 71

Prevention of Contamination in PCR Laboratory:

Prevention 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. Dr.T.V.Rao MD 72

Polymerase Chain Reaction Available for several infections ….:

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 for corneal specimens Dr.T.V.Rao MD 73

Uses and Advantages in Testing by PCR Methods:

Uses and Advantages in Testing by PCR Methods Clinical diagnostics: detection and quantification of infectious microorganisms, cancer cells and genetic 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 of sample contamination Amplifies products from a wide variety of total RNA or mRNA sources Dr.T.V.Rao MD 74

Disadvantages of PCR Methods:

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 Dr.T.V.Rao MD 75

Molecular Epidemiology in Eye Care:

Molecular Epidemiology in Eye Care In several world health funded projects molecular methods are used for 1 Plasmid analysis 2 Genomic fingerprinting 3 Emerging drug resistance 4 Polymerase chain reaction to identify emerging and remerging microbes, 5 Determination of antibiotic resistance patterns. Dr.T.V.Rao MD 76

Be Familiar with Molecular Biology or ???:

Be Familiar with Molecular Biology or ??? Dr.T.V.Rao MD 77

Bioinformatics may take over Biology ???:

Bioinformatics may take over Biology ??? Dr.T.V.Rao MD 78

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Programme created by Dr.T.V.Rao MD for basic awareness on Molecular Methods in Diagnosis Email doctortvrao@gmail.com Dr.T.V.Rao MD 79

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