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Dr.T.V.Rao MD
Dr.T.V.Rao MD
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
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Dr.T.V.Rao MD
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
Important Methods
1 Nucleic acid probes 2 Hybrid capture 3 Branched chain DNA 4 Situ hybridization
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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."
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Hybridization probe
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 probetarget base pairing due to complementarily between the probe and target.
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The rungs of the ladder can occur in any order (as long as the basepair rule is followed) Those 4 bases have endless combinations just like the letters of the alphabet can combine to make different words.
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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.
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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
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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.
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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
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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 .
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Western Blotting
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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
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Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA.
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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.
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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.
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Spoligotyping in Tuberculosis
The well-conserved 36bp 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.
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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
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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 )
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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.
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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.
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The dreaded infections endophthalmitis following cataract extraction and lens implantation often are caused by opportunistic pathogens
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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.
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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.
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Every organism contains some unique, species specific DNA sequences Molecular diagnostics makes the species specific DNA visible
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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
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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.
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Dr.T.V.Rao MD
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.
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KirkBloods worth case A Waterman Imprisoned for 9 years on wrong evidences of Rape Unmatched DNA by PCR makes a freeman
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Dr.T.V.Rao MD
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.
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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 colourblindness than women.
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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.
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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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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.
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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.
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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
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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.
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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
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Paste Amplify
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PCR Primers
TTAACGGCCTTAA . . . TTTAAACCGGTT AATTGCCGGAATT . . . . . . . . . .> and <. . . . . . . . . . AAATTTGGCCAA TTAACGGCCTTAA . . . TTTAAACCGGTT
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Denaturing Template
Heat causes DNA strands to separate
5 3 3 5
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Annealing Primers
Primers bind to the template sequence
3 5
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3
3
5
Extend 72oC
5 3
5 3 3 5
3 5
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Applications of PCR
The standard specimen procedure can quantitate HIV1 RNA in a range of 40075,000 copies/mL.
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Advantages of PCR
Speed
Ease
of use Sensitivity
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fragments of 125-bp (EO3) and 317 bp (HSP) specific for C. albicans were used for amplification.
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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
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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.
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High sensitivity and specificity Detects pathogen, not immune response Quick results High transport toleration
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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.
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RobusT RT-PCR Kits perform cDNA synthesis and PCR amplification of cDNA successively in a single tube during a continuous thermal cycling
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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
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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
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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.
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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.
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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
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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
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.
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Programme created by Dr.T.V.Rao MD for basic awareness on Molecular Methods in Diagnosis Email doctortvrao@gmail.com
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