Recombinant DNA Technology- Part 1.pdf
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Recombinant DNA technology involves manipulating DNA sequences in the laboratory. DNA is isolated, cut with restriction enzymes, and joined with DNA ligase. The recombinant DNA is inserted into a cloning vector and introduced into a host cell. Cells containing the recombinant DNA are selected and amplified. This allows large quantities of identical DNA molecules to be produced for analysis, comparison, and other purposes. Common applications include understanding disease, producing therapeutic proteins, disease prevention through vaccines, diagnosis, and gene therapy.
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Recombinant DNA Technology- Part 1.pdf
- 1. Recombinant DNA Technology part 1 NAMRATA CHHABRA
- 2. What is Recombinant DNA ? ● Recombinant DNA, also known as Chimeric DNA ● Contains sequences taken from very different sources, ● e.g. DNA containing both human and bacterial DNA sequences Pig-monkey hybrids are born alive in China
- 3. Recombinant DNA Technology ● Laboratory manipulation of the inherited characteristics of cells and organisms. The technology involves: ● Excising ● Joining, and ● Cloning specific sequences of DNA
- 4. Genetic engineering Manipulation of a DNA sequence and the construction of chimeric molecule is also called genetic engineering.
- 5. Purpose of Recombinant DNA technology This is the laboratory manipulation of nucleic acids to- • Analyze • Compare • Construct • Mutate
- 6. Purpose of genetic engineering (1) Understanding the molecular basis of diseases. For example, ● familial hypercholesterolemia, ● sickle-cell disease, ● thalassemias, ● cystic fibrosis, ● muscular dystrophy, ● vascular and heart disease, ● Alzheimer disease, cancer, obesity and diabetes.
- 7. Purpose of genetic engineering (2) Synthesis of human proteins in abundance for therapeutic purpose eg, ● Insulin, ● Growth hormone, ● Tissue plasminogen activator ● Vaccines etc.
- 8. Purpose of genetic engineering (3) Disease prevention -vaccines (eg, hepatitis B) (4) Diagnosis- for diagnostic testing (eg, Ebola and AIDS tests) (4) Risk prediction of developing a given disease and (5) Prediction of response to pharmacological therapeutics.
- 9. Purpose of genetic engineering (5) Crime detection- DNA fingerprinting, remarkable advances in forensic medicine. (6) Gene therapy for potentially curing diseases caused by a single-gene deficiency such as sickle-cell disease, the thalassemias, adenosine deaminase deficiency, and others may be devised.
- 10. Steps involved in Recombinant DNA Technology The procedure involves: 1) Isolation of DNA 2) Cutting DNA at precise locations 3) Joining two DNA fragments covalently 4) Selection of a small molecule of DNA capable of self- replication 5) Moving recombinant molecules from the test tube in to a host cell 6) Selecting or identifying those cells that contain Recombinant DNA
- 11. 1) Isolation of DNA ● The first step involves isolating the desired DNA in its pure form i.e. free from other macromolecules. ● Since DNA exists within the cell membrane along with other macromolecules such as RNA, polysaccharides, proteins, and lipids, it must be separated and purified.
- 12. 2) Cleavage of DNA ● Special enzymes, Restriction enzymes cut DNA chains at specific locations. ● These enzymes are also called “Molecular scissors” ● The specific site is called “Restriction site” which is 4-7 base pair long ● The fragments of DNA obtained after the action of restriction enzymes are called “Restriction fragments”; they can have sticky or blunt ends. ●
- 13. Restriction endonucleases ● These enzymes were called restriction enzymes because their presence in a given bacterium restricted (ie, prevented) the growth of certain bacterial viruses called bacteriophages. ● Endonucleases—enzymes that cut DNA at specific DNA sequences within the molecule (as opposed to exonucleases,which processively digest from the ends of DNA molecules)
- 14. Restriction endonucleases How is the host DNA protected? Restriction endonucleases are present only in cells that also have a companion enzyme that site-specifically methylates the host DNA, rendering it an unsuitable substrate for digestion by that particular restriction enzyme. Thus, site-specific DNA methylases and restriction enzymes that target the exact same sites always exist in pairs in a bacterium.
- 15. Site specific cleavage ● Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is typically 4 to 7 bp long (restriction site). ● Most recognition sequences are palindromes (ie, the sequence reads the same in opposite directions on the two strands). ● By convention, these are written in the 5′ to 3′ direction for the upper strand of each recognition sequence, and the lower strand is shown with the opposite (ie, 3′-5′) polarity.
- 16. Nomenclature of Restriction endonucleases ● Restriction enzymes are named after the bacterium from which they are isolated. ● For example, EcoRI is from Escherichia coli, and BamHI is from Bacillus amyloliquefaciens. ● The first three letters in the restriction enzyme name consist of the first letter of the genus (E) and the first two letters of the species (co). ● These may be followed by a strain designation (R) and a roman numeral (I) to indicate the order of discovery (eg, EcoRI and EcoRII)
- 17. Restriction fragments These DNA cuts result in blunt ends (eg, HpaI) or overlapping (sticky or cohesive) ends (eg, BamHI) depending on the mechanism used by the enzyme.
- 18. Sticky ends ● Sticky, or complementary cohesive-end ligation of DNA fragments is technically easy. ● They are particularly useful in constructing hybrid or chimeric DNA molecules ● However, sticky ends of a vector may reconnect with themselves, with no net gain of DNA. ● Sticky ends of fragments also anneal so that heterogeneous tandem inserts form. ● Also, sticky-end sites may not be available or in a convenient position
- 20. Blunt ends To circumvent these problems, an enzyme that generates blunt ends can be used. Blunt ends can be ligated directly; however, ligation is not directional. Two alternatives thus exist: Homopolymer tailing New ends are added using the enzyme terminal transferase or synthetic sticky ends are added. If poly d(G) is added to the 3′ ends of the vector and poly d(C) is added to the 3′ ends of the foreign DNA using terminal transferase, the two molecules can only anneal to each other, thus circumventing the problems listed above.
- 21. Blunt ends Alternatively, synthetic blunt-ended duplex oligonucleotide linkers containing the recognition sequence for a convenient restriction enzyme sequence are ligated to the blunt-ended DNA. Direct blunt-end ligation is accomplished using the bacteriophage T4 enzyme DNA ligase. This technique, though less efficient than sticky-end ligation, has the advantage of joining together any pairs of ends.
- 23. A revision of mechanism of action of restriction endonuclease
- 24. 2) Joining two DNA fragments covalently The DNA fragments are joined together by DNA ligase
- 25. DNA Ligase In DNA replication, ligase’s function is to join together fragments of newly synthesized DNA to form a seamless strand. The ligases used in DNA cloning do basically the same thing. If two pieces of DNA have matching ends, DNA ligase can join them together to make an unbroken molecule.
- 26. DNA Ligase ● Using ATP as an energy source, ligase catalyzes a reaction in which the phosphate group sticking off the 5’ end of one DNA strand is linked to the hydroxyl group sticking off the 3’ end of the other. ● This reaction produces an intact sugar-phosphate backbone.
- 27. Example- Use of restriction endonuclease and ligase
- 28. Example- Use of restriction endonuclease and ligase
- 29. Example- Use of restriction endonuclease and ligase
- 31. III) Cloning A clone is a large population of identical molecules, bacteria, or cells that arise from a common ancestor. Molecular cloning allows for the production of a large number of identical DNA molecules, which can then be characterized or used for other purposes.
- 32. Cloning vectors ● Chimeric or hybrid DNA molecules can be inserted in cloning vectors which then continue to replicate in a host cell under their own control systems. ● In this way, the chimeric DNA is amplified.
- 33. Cloning vectors A vector is a molecule of DNA to which the fragment of DNA to be cloned is attached. Commonly used vectors are: • Plasmids • Bacterial and animal viruses • Cosmids • Artificial chromosomes
- 34. Cloning vectors
- 35. Bacterial Plasmids ● Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to the host cell. ● They exist as single or multiple copies within the bacterium and replicate independently from the bacterial DNA ● Plasmids have several properties that make them extremely useful as cloning vectors.
- 36. Plasmids 1. Origin of replication 2. Site for restriction endonucleases 3. Identification markers (antibiotic resistance genes)
- 37. Plasmids ● Plasmids range in size from a few thousand base pairs to more than 100 kilobases (kb). ● Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division. ● During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell.
- 38. Plasmids as cloning vectors ● The plasmids most commonly used in recombinant DNA technology replicate in E. coli. ● Generally, these plasmids have been engineered to optimize their use as vectors in DNA cloning. ● For instance, to simplify working with plasmids, their length is reduced; many plasmid vectors are only ≈3kb in length, which is much shorter than in naturally occurring E. coli plasmids.
- 39. Bacterial and animal viruses ● Phages usually have linear DNA molecules into which foreign DNA can be inserted at several restriction enzyme sites. ● A major advantage of phage vectors is that while plasmids accept DNA pieces about 6– 10 kb long, phages can accept longer DNA fragments.
- 40. Bacteriophage- a cloning vector
- 41. Cosmids as cloning vectors ● Larger fragments of DNA can be cloned in cosmids ● Combine the best features of plasmids and phages. ● Contain the DNA sequences, so-called cos sites, required for packaging lambda DNA into the phage particle. ● These vectors grow in the plasmid form in bacteria, ● Chimeric DNA can be packaged into the particle head. ● Cosmids can carry inserts of chimeric DNA that are 35 to 50 kb long.
- 42. Cosmids
- 43. Artificial Chromosomes Even larger pieces of DNA can be incorporated into bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC). These vectors can accept and propagate DNA inserts of several hundred kilobases or more and have largely replaced the plasmid, phage, and cosmid vectors.
- 44. Yeast artificial chromosome YACs contain selection, replication, and segregation functions that work in both bacteria and yeast cells and therefore can be propagated in either organism.
- 45. Viral vectors Mammalian viral vectors are also used in this technology especially for gene therapy.
- 46. 4) Moving recombinant molecules from the test tube into a host cell E. coli is the most common host cell due to the following advantages: i) It’s DNA metabolism is well understood ii) Cloning vectors associated with E.Coli are well characterized iii)Effective techniques are available for moving DNA from one bacterial cell to another
- 47. Transformation ● The term transformation is used to denote the genetic alteration of a cell caused by the uptake and expression of foreign DNA regardless of the mechanism involved. ● The phenomenon of transformation permits plasmid vectors to be introduced into and expressed by E. coli cells. ● Normal E. coli cells cannot take up plasmid DNA from the medium. ● Exposure of cells to high concentrations of certain divalent cations, however, makes a small fraction of cells permeable to foreign DNA by a mechanism that is not understood.
- 48. Transformation ● In a typical procedure, E. coli cells are treated with CaCl2 and mixed with plasmid vectors; ● commonly, only 1 cell in about 10,000 or more cells becomes competent to take up the foreign DNA. ● Transformation can also be undertaken by giving a heat shock
- 49. 5) Selecting or identifying those cells that contain Recombinant DNA ● Colony or plaque hybridization is the method by which specific clones are identified and purified. ● Bacteria are grown as colonies on an agar plate.
- 50. Colony or plaque hybridization Colonies are overlaid with nitrocellulose filter paper. Cells from each colony stick to the filter and are permanently fixed thereto by heat, which with NaOH treatment also lyses the cells and denatures the DNA so that it will hybridize with the probe. https://www.youtube.com/watch? v=PEwWXrRWvWE
- 51. Selecting or identifying those cells that contain Recombinant DNA ● A radioactive probe is added to the filter, and (after washing) the hybrid complex is localized by exposing the filter to x-ray film. ● By matching the spot on the autoradiograph to a colony, the latter can be picked from the plate. ● A similar strategy is used to identify fragments in phage libraries. ● Successive rounds of this procedure result in a clonal isolate (bacterial colony) or individual phage plaque.
- 52. Colony or plaque hybridization
- 53. Viral vectors ● Adenoviral (Ad), ● Adenovirus associated viral (AAV) (DNA-based) and ● Retroviral (RNA based) genomes. ● Though somewhat limited in the size of DNA sequences that can be inserted, such mammalian viral cloning vectors make up for this shortcoming because they will efficiently infect a wide range of different cell types.
- 54. Gene therapy
- 55. DNA library ● Each colony is a cell clone, but it is also a DNA clone because the recombinant vector has now been amplified by replication during every round of cell division. ● Thus, the Petri dish, which may contain many hundreds of distinct colonies, represents a large number of clones of different DNA fragments. ● This collection of clones is called a DNA library.
- 56. Genomic library ● A genomic library is prepared from the total DNA of a cell line or tissue. ● Genomic DNA libraries are often prepared by performing partial digestion of total DNA with a restriction enzyme that cuts DNA frequently (eg, a four base cutter such as TaqI). ● The idea is to generate rather large fragments so that most genes will be left intact. ● The BAC, YAC, and P1 vectors are preferred since they can accept very large fragments of DNA
- 57. Genomic library By considering the size of the donor genome and the average size of the inserts in the recombinant DNA molecule, a researcher can calculate the number of clones needed to encompass the entire donor genome, or, in other words, the number of clones needed to constitute a genomic library.
- 58. cDNA library ● Creation of a cDNA library begins with messenger ribonucleic acid (mRNA) instead of DNA. ● Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. ● To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA. ● The resulting DNA molecules are called complementary DNA (cDNA). ● A cDNA library represents a sampling of the transcribed genes, whereas a genomic library includes untranscribed regions.
- 59. cDNA library A cDNA library comprises complementary DNA copies of the population of mRNAs in a tissue. It represents a collection of only the genes that are encoded into proteins by an organism.
- 60. Expression vectors These vectors are specially constructed to contain: ● very active inducible promoters, proper in- phase translation initiation codons,both transcription and translation termination signals, and appropriate protein processing signals, if needed. ● Some expression vectors even contain genes that code for protease inhibitors, so that the final yield of product is enhanced.
- 62. Blot transfer Visualization of a specific DNA or RNA fragment among the many thousands of “contaminating” non-target molecules in a complex sample requires the convergence of a number of techniques, collectively termed blot transfer. ● Southern (DNA), ● Northern (RNA), and ● Western (protein) blot transfer procedures.
- 63. Southern hybridization It is named for the person who devised the technique [Edward Southern], and the other names began as laboratory jargon but are now accepted terms
- 65. Steps of southern hybridization Step I: Restriction digestion 1. DNA is extracted from the cell. 2. It is partially digested by a restriction endonuclease (RE) that cuts the DNA at a specific site generating fragments (obtaining complete fragmentation of DNA at the intended restriction enzyme sites is a critical step in Southern blot analysis). 3. The fragments of DNA obtained by restriction digestion are amplified by PCR.
- 66. Steps of southern hybridization Step II: Gel electrophoresis 4. The resulting DNA fragments are separated by electrophoresis. Fragmented DNA is typically electrophoresed on an agarose gel to separate the fragments according to their molecular weights. Acrylamide gels can alternatively be used for good resolution of smaller DNA fragments (<800 bp). Step III: Denaturation 5. The gel after electrophoresis is then soaked in alkali (NaOH) or acid (HCl) to denature the double-stranded DNA fragments.
- 67. Steps of southern hybridization Step IV: Blotting 6. Without altering their positions, the separated bands of ssDNA are transferred to a nitrocellulose filter by the process of blotting. Step V: Baking and blocking 7. After the DNA of interest is bound on the membrane, it is baked on autoclave to fix in the membrane. The membrane is then treated with casein or bovine serum albumin (BSA) which saturates all the binding site of membrane
- 68. Steps of southern hybridization Step VI: Hybridization with labeled probes 8. The DNA bound to membrane is then treated with labeled probe. The probe binds with complementary DNA on the membrane since all other nonspecific binding sites on the membrane are blocked by BSA or casein. The probe can be labeled with radioactivity, fluorescent dye, or an enzyme that can generate a chemiluminescent signal when incubated with the appropriate substrate.
- 69. Steps of southern hybridization Step VII: Visualization by Autoradiogram 9. The membrane bound DNA labeled with probe can be visualized under autoradiogram which give pattern of bands. 10. In the detection step, the bound, labeled probe is detected using the method required for the particular label used. For example, radiolabeled probes may be detected using X-ray film or a phosphor imaging instrument, and enzymatically labeled probes are typically detected by incubating with a chemiluminescent substrate and exposing the blot to X-ray film.
- 70. Applications of Southern blot DNA fingerprinting is an example of southern blotting. ● Used for paternity testing, criminal identification, and victim identification. ● To identify mutation or gene rearrangement in the sequence of DNA. ● Used in diagnosis of diseases caused by genetic defects. ● Used to identify infectious agents.
- 71. Applications of Southern blot ● Identification of one or more restriction fragments that contain a gene or other DNA sequence of interest and ● in the detection of RFLPs used in construction of genomic maps.
- 72. Restriction fragment length polymorphism ● Treatment of genomic DNA from different individuals with a single restriction enzyme does not always give the same set of fragments ● because some restriction sites are polymorphic, ● being present in some individuals but absent in others, ● usually because a point change in the nucleotide sequence changes the restriction site into a sequence not recognized by the restriction enzyme
- 73. Restriction fragment length polymorphism
- 74. Northern blotting The northern blot is a technique used in molecular biology to study gene expression by detection of RNA (or isolated mRNA) in a sample. Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence. They can be DNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence.
- 76. Western blotting ● The western blot (also called the immunoblot) is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. ● It uses gel electrophoresis to separate native proteins by 3D structure or denatured proteins by the length of the polypeptide. ● The proteins are then transferred to a membrane (typically nitrocellulose), where they are hybridized with antibodies specific to the target protein.
- 77. Western Blotting
- 78. Polymerase chain reaction (PCR) • A molecular technique to copy or amplify small segments of DNA or RNA. • An efficient and cost-effective technique that combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication that are applied repeatedly through numerous cycles. • It results in the exponential production of the specific target DNA/RNA sequences by a factor of 107 within a relatively short period.
- 79. Components of Polymerase Chain Reactions (PCR) DNA template (the sample DNA that contains the target sequence to amplify) • Deoxyribonucleoside triphosphates (dNTPs) • PCR buffer • Primers (forward and reverse) • Taq polymerase
- 80. Steps of procedure • The PCR amplification occurs by repeated cycles of three temperature-dependent steps called : • denaturation, • annealing, and • elongation
- 81. Factors affecting PCR Primer • A short segment of nucleotides (15-30 bases long) which is complementary to a section of the DNA or RNA, which is to be amplified in the PCR. • Two short DNA sequences designed to bind to the start (forward primer) and end (reverse primer) of the target sequence are used in PCR.
- 82. Forward and reverse primers The primer that anneals with the antisense strand or the noncoding strand or the template strand is known as forward primer. Reverse primer is the short DNA sequence that anneals with the 3’ end of the sense strand or the coding strand.
- 83. Factors affecting PCR Primer (contd.) • Both reverse and forward primers are important for the production of millions to billions of copies of particular regions of DNA that are targeted or interested. • Hence optimal concentration of primer is needed • Low concentration of primer results in poor yield while high concentration may result in non-specific amplification.
- 84. Factors affecting PCR ii. Amount of Template DNA • Optimal amount of template DNA usually is in nanogram. • Higher concentrations inhibit or result in non -specific amplification.
- 85. Factors affecting PCR • iii) Taq DNA polymerase: • A heat-stable DNA polymerase obtained from the thermophilic bacterium, Thermus. aquaticus is used to synthesize the new strands in most PCR reactions. • Since this Taq DNA polymerase works best at around 72°C (161.6°F), the temperature of the PCR mixture is raised to that temperature for elongation to proceed efficiently. • Taq DNA polymerase has both 5’-3’ polymerase and 5’-3’ exonuclease activities. But it lacks 3’-5’ exonuclease activity (proofreading activity).
- 86. Detection of PCR products • Labeled probe that is specific for the target gene sequence is used to detect PCR amplified gene product (also known as amplicon). • Based on the nature of the reporter molecule used, probe generates radioactive, colorimetric, fluorometric, or chemiluminescent signals.
- 87. Steps of procedure • The PCR is carried out in a single test tube containing all the necessary components. • The extracted sample (which contains target DNA template) is added to the tube containing primers, free nucleotides (dNTPs), and Taq polymerase. • The PCR mixture is placed in a PCR machine, that increases and decreases the temperature of the PCR mixture in automatic, programmed steps and the copies of the target sequence are generated exponentially.
- 88. Steps of procedure 1) Denaturation (strand separation): • Native DNA exists as a double helix • Denaturation separates the two DNA chains by • heating the reaction mixture to 90°C to 95°C (194°F to 203°F).
- 89. Steps of procedure 2) Annealing (primer binding): • the reaction mixture is cooled to 45°C to 60°C (113°F to 140°F) • so that the oligonucleotide primers can bind or anneal to the separated strands of the target DNA.
- 90. Steps of procedure 3) Extension (synthesis of new DNA): During elongation, the DNA polymerase adds nucleotides to the 3 ‘ends of the primers to complete a copy of the target DNA template. These three steps are repeated 20-30 times in an automated thermocycler that can heat and cool the reaction mixture in tube within very short time. This results in exponential accumulation of specific DNA fragments.
- 91. Steps of procedure • PCR is a method to amplify any DNA sequence virtually without limit and allows the separation of the nucleic acid of interest from its context. • The doubling of number of DNA strands corresponding to target sequences can be estimated by amplification number associated with each cycle using the formula. • Amplification=2n, where n=no. of PCR cycle
- 93. Reverse transcription PCR • To carry out polymerase chain reaction where RNA is the starting material this method uses reverse transcriptase, a process called RT– PCR (reverse transcriptase polymerase chain reaction). • The first step in this method is to convert the RNA molecules into single- stranded complementary DNA (cDNA). • After this step, the experiment proceeds as in the standard technique.
- 94. RT PCR
- 96. PCR • PCR can amplify a desired DNA sequences of any origin hundred or millions time in a matter of hour, which is very short in comparison to recombinant DNA technology. • PCR is especially valuable because the reaction is highly specific, easily automated and very sensitive. • It is widely used in the fields like- clinical medicine for medical diagnosis, diagnosis of genetic diseases, forensic science; DNA fingerprinting, evolutionary biology.
- 97. Applications of PCR 1.Forensic science: DNA fingerprinting, paternity testing and criminal identification
- 100. Applications of PCR 2.Diagnosis: Identification and characterization of infectious agents • Direct detection of microorganisms in patient specimens • Identification of microorganisms grown in culture • Detection of antimicrobial resistance • Investigation of strain relatedness of pathogen of interest
- 101. Diagnostic significance of PCR • PCR is becoming the leading method for detection of the continuously increasing number of human pathogens. • Examples include: • Herpes simplex virus (HSV), Human papillomavirus, Human immunodeficiency virus (HIV), Human T lymphotropic virus type I and type II, • Cytomegalovirus (CMV), • Epstein-Barr virus, • Human herpesvirus-6 (HHV-6), • Hepatitis B virus, rubella virus, mycobacteria, Toxoplasmosis gondii, Trypanosoma cruzi, and malaria.
- 102. Applications of PCR 3. Detection of mutation (investigations of genetic diseases)
- 103. Applications of PCR 8. Vaccine production by recombinant DNA technology
- 104. Applications of PCR 9. Drug discovery 10. Human genome project
- 105. Applications of PCR 6. Gene cloning and expression
- 106. Applications of PCR 7. Gene sequencing
- 107. Applications of PCR 4. Evolution study: evolutionary biology 5. Fossil study: paleontology
- 108. Advantages of PCR over other diagnostic procedures 1) Sensitivity and specificity- PCR can be applied as a detection method for virtually any pathogen for which even limited nucleotide sequence information is known and for which even a very small amount of a specimen of infected tissue is available. 2) Discriminative- In most cases PCR assays are more discriminative than conventional serology. For example, it is difficult to distinguish HSV-I from HSV-II or HIV-1 from HIV-2 by serology, yet such distinctions can be readily made based on PCR amplification of type-specific genetic sequences.
- 109. Advantages of PCR over other diagnostic procedures 3) PCR yields rapid results, typically in 1 to 2 days in a clinical setting. The results are specific, faster and accurate 4) It is applicable to a wide variety of clinical, pathologic, or forensic specimens, as well as to formalin-fixed tissue, inactivated bacterial cultures, and archaeological specimens.
- 110. Summary 1) It’s an efficient and cost-effective technique that combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication that are applied repeatedly through numerous cycles. 2) The PCR amplification occurs by repeated cycles of three temperature- dependent steps called denaturation, annealing, and elongation. 3) Labeled probe that is specific for the target gene sequence is used to detect PCR amplified gene product (also known as amplicon).
- 111. Summary 4) PCR can be applied as a detection method for virtually any pathogen for which even limited nucleotide sequence information is known and for which even a very small amount of a specimen of infected tissue is available. 5) PCR yields rapid results, typically in 1 to 2 days in a clinical setting. The results are specific, faster and accurate
- 112. Thank you