Polymerase Chain Reaction Technology and its applications he enormous advances made in our understanding of the human genome (and that of many other species), would not have been possible, were it not for the remarkable simple and yet exquisitely adaptable technique which is PCR. It involves the principle of reactions as - PCR represents a cyclic reaction where target DNA is amplified in vitro by
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a series of polymerization cycles. Each cycle includes three steps: • Heating step at 910–970C for 20–30 seconds, where the DNA template duplex is denatured to single strands. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases,
yielding single strands of DNA. For multiple copies of DNA molecules, the melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the “random-coil” states. The melting temperature depends on both the length of the molecule, and the May-June 2010 Medical Equipment & Automation
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Forensic Technology Polymerase chain reaction (PCR) is a technique widely used in molecular biology, biotechnology, microbiology, genetics, diagnostics, clinical laboratories, forensic science, environmental science, hereditary studies, paternity testing, and many other applications. It is a primer-mediated enzymatic amplification process of specifically cloned or genomic DNA sequences, which was invented by Dr. Kary Banks Mullis who received a Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR). This process, which Kary conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century. It is a method of amplifying DNA; PCR multiplies a single, microscopic strand of the genetic material billions of times within hours. This technology has been developed in areas as diverse as criminal forensic investigations, food science, ecological field studies, and diagnostic medicine. specific nucleotide sequence composition of that molecule. • Annealing step usually at 400–650C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-50C below the Tm of the primers used. Stable DNADNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis, and • Extension step at 680–730C where thermostable DNA polymerase catalyzes the synthesis of a new DNA strand by elongation of the primed strand. The reaction requires two short oligonucleotides (primers) flanking the target region to be amplified, which are present in large molar excess and hybridize to complementary segments of DNA. During the reaction, deoxynucleotide triphosphates (dNTP), i.e.,
dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate) and dTTP (deoxythymidine triphosphate), are bound to the free 3’-hydroxyl end of the new strand. Only deoxynucleotide monophosphate is incorporated in the DNA chain, cleaving off a pyrophasphate group. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified (Fig. 1).
temperature step (called hold) at a high temperature (>900C), and followed by one hold at the end for final product extension or brief storage. The temperatures
The PCR usually consists of a series of 20-40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete Fig. 1: Showing three fundamental steps of PCR cycle temperature steps. Most commonly PCR is carried used and the length of time they out with cycles that have three are applied in each cycle depend temperature steps. The cycling on a variety of parameters. These is often preceded by a single
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Forensic Technology include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs (deoxynucleoside triphosphates) in the reaction, and the melting temperature (Tm) of the primers. The target copies are doublestranded and bounded by annealing sites of the incorporated primers. The 3’ end of the primer should complement the target exactly, but the 5’ end can actually be a non-complementary tail with restriction enzyme and promoter sites that will also be incorporated. As the cycles proceed, both the original template and the amplified targets serve as substrates for the denaturation, primer annealing, and primer extension processes. Since every cycle theoretically doubles the amount of target copies, a geometric amplification occurs. PCR is conceptualized as a process that begins when thermal cycling ensues. The annealing temperature sets the specificity of the reaction, assuring that the primary primer binding events are the ones specific for the target in question. In preparing a PCR reaction on ice or at room temperature, however, the reactants are all present for nonspecific primer annealing to any single-stranded DNA present. Since Taq DNA polymerase has some residual activity even at lower temperatures, it is possible to extend these misprimed hybrids and begin the PCR process at the wrong sites. By withholding a key reaction component, such as Taq DNA polymerase, until an elevated temperature can be reached, the possibility of mispriming is avoided. This can be accomplished by a manual addition of enzyme
above 65—700C during the first heating ramp to denaturation at 940C. Alternatively, an inactive form of the enzyme AmpliTaq Gold can be added to all reactions to prevent misprimed extensions. Adding a pre-PCR heat step at 92-950C for 9-12 min synchronously reactivates the enzyme and achieves an “invisible” hot start. In both cases, the lowest temperature experienced by the reaction components is the stringent primer annealing temperature, assuring best specificity.
PCR must be performed in vessels that are compatible with low amounts of enzyme and nucleic acids and that have good thermal transfer characteristics. Typically, polypropylene is used for PCR vessels and conventional, thick-walled microcentrifuge tubes are chosen for many thermal cycler systems. PCR is most often performed at a 10-100 μL reaction scale and requires the prevention of the evaporation/condensation processes in the closed reaction tube during thermal cycling.
AmpliTaq DNA Polymerase is a highly characterized recombinant enzyme for PCR. It is produced in E. coli from the Taq DNA polymerase gene, thereby assuring high purity. It is commonly supplied and used as a 5 U/μL solution in buffered 50% glycerol.
Mineral oil overlay or wax layer serves this purpose. More recently, 0.2 mL thin-walled vessels have been optimized for the PCR process and oil-free thermal cyclers have been designed that use a heated cover over the tubes held within the sample block. In a standard aliquot of Taq DNA polymerase used for a 100μL reaction, there are about 1010 molecules. Each PCR sample should be evaluated for the number of target copies it contains or may contain. For example 1ng of λ DNA contains 1.8 x 107 copies. For low input copy number PCR, the enzyme is in great excess in early cycles.
The enzyme is a 94-kDa protein with a 5’-3’ polymerization activity that is most efficient in the 70800C range. This enzyme is very thermostable, with a half-life at 950C of 35-40 min. AmpliTaq DNA polymerase requires magnesium ion as a cofactor and catalyzes the extension reaction of a primed template at 720C. As mentioned above, the four dNTPs (consisting of dATP, dCTP, dGTP, and dTTP or dUTP) are used according to the base-pairing rule to extend the primer and thereby to copy the target sequence. Modified nucleotides (ddNTPs, biotin-11 -dNTP, dUTP, deaza-dGTP, and fluorescently labeled dNTPs) can be incorporated into PCR products. The PCR buffer for Taq DNA polymerase consists of 50 mM KC1 and 10 mM Tris-HCl, pH 8.3, at room temperature. This buffer provides the ionic strength and buffering capacity needed during the reaction.
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As the amplicon accumulates in later cycles, the enzyme becomes limiting and it may be necessary to give the extension process incrementally more time. Thermal cyclers can reliably perform this automatic segment extension procedure in order to maximize PCR yield. The Polymerase Chain Reaction (PCR) Technology can be applied in various forms needing small modification to be made to the standard PCR protocol to achieve a desired goal. The prominent categories with their applications are described below. May-June 2010 Medical Equipment & Automation
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Forensic Technology PCR Analysis of DNA from Fresh and Decomposed Bodies and Skeletal Remains in Medico-legal Death Investigations One of the greatest values of polymerase chain reaction (PCR) for the death investigator lies in the fact that even minute amounts of DNA or extensively damaged (degraded) DNA can be successfully amplified and thus become amenable for typing procedures. In medico-legal death investigations, the types of DNA recovered from a body can be divided into two areas: DNA evidence, which adheres to the surface of the body or is present within a body cavity (e.g., blood, semen, saliva, nasal secretions, hairs, shed scalp skin, urine, faeces); and biological material, which belongs to the deceased (e.g., liquid blood, soft tissues, bones, teeth, fingernails) and can be used as reference samples. In general, the success of DNA profiling depends most on the environmental conditions the body was exposed to and on the proper preservation and collecting procedures.
PCR Analysis from Cigarette Butts, Postage Stamps, Envelope Sealing Flaps, and Other SalivaStained Material The polymerase chain reaction (PCR) has offered the forensic scientist a new range of sensitivity in the examination of forensic samples. PCR has been successfully used to amplify specific DNA fragments from extremely small amounts of DNA present on cigarette butts, postage stamps, envelope sealing flaps, and other saliva-stained materials. In addition to DNA typing results, it is at times desirable to confirm the presence of saliva by
the simultaneous employment of an amylase assay.
Mutation and Polymorphism Detection Mutational analysis has many applications, for example, markers of disease, cancer research, and genotyping. Mutations are responsible for diseases, such as sickle cell anemia, cystic fibrosis, and adrenal leukodystrophy. An expansion of the CAG repeat in exon 1 of the androgen receptor alone can result in Kennedy’s disease, spinocerebellar ataxia, or fragile X syndrome. Therefore, polymorphism and mutational analysis are of use in diagnosing these diseases. Detection of polymorphism and mutational analysis is also widely used in the field of cancer research. Mutation detection has demonstrated that p53 function is lost in approximately 50% of all cancers and that the loss of function is caused by point mutations. It also demonstrates that a mutation in BRCA 1 or BRCA 2 increases susceptibility to breast and ovarian cancer. Microsatellite analysis is used in cancer research to study LOH (loss of heterozygosity) and microsatellite instability. LOH studies have demonstrated most genetic alterations that occur in bladder cancer are on chromosome 9 and that microsatellite instability is commonly found in colorectal cancer. In summary, polymorphism and mutational analysis is a major tool in science today, without which much of our current understanding of genetics would not have been possible.
Ultrasensitive Quantitative PCR to Detect RNA Viruses
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polymerase chain reaction (qPCR) to detect RNA viruses has become increasingly important as a prognostic marker and in patient management, for example, in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. Drug therapies can be monitored by regularly checking viral load, indicating whether the regime is sufficient, or whether alternatives should be sought. It is therefore crucial that the systems used are ultrasensitive and give accurate and reproducible results.
Single-Locus and Multilocus VNTR-PCR Nonisotopic probes have been widely adopted for DNA fingerprinting and DNA profiling because of their ease and speed of use and obvious safety and environmental advantages. Nonisotopic DNA probes designed to detect variable number of tandemrepeat (VNTR) sequences are typically single-stranded oligomers of 20-30 nucleotides, with sequence complementary to the target tandem-repeat sequence. There are two types of probes used for the analysis of VNTR sequences: multilocus probes (MLP) and singlelocus probes (SLP). MLPs consist of tandem repeats containing a minisatellite “core” sequence that can simultaneously detect a number of highly polymorphic loci to generate individual-specific DNA “fingerprints”. These probes have found a number of applications in the field of identity analysis, including forensic and paternity testing and cell-line verification. When hybridized to Southern blots under conditions of low stringency, each multilocus probe will detect a family of minisatellites that May-June 2010 Medical Equipment & Automation
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Forensic Technology all share the same “core” sequence. This produces the multiband DNA “fingerprint” pattern. Several different MLPs have been isolated, and most can be used to detect minisatellites in a wide range of species. PCR-VNTRs are important markers for questions of identification, individualization, and discrimination. They already form an integral part of forensic DNA analysis.
Direct and Indirect In Situ PCR In recent years, the development of in situ technologies has made good progress. In situ hybridization (ISH) has become an important tool and has enabled the pathologist to demonstrate infectious pathogens or mRNAs in tissue sections or cytospins without destruction of morphology, thus enabling the assignment of signals to individual cells or cell compartments.
mtDNA existing in hundreds if not thousands of copies per cell. The discriminatory power of mtDNA testing arises from the polymorphic nature (between unrelated individuals) of the two hypervariable regions (HV1 and HV2) located within the D-loop of the mtDNA genome. The haploid, maternal inheritance patterns of mtDNA transmission between generations, allow an inclusion or exclusion to be made when the sample sequence is compared to that of a maternal reference. Related individuals will share similar polymorphisms relative to a consensus standard.
Reverse-transcriptasepolymerase chain reaction (RT-PCR) in Biomedicine
Amplification and Sequencing of Mitochondrial DNA in Forensic Casework
RT-PCR has become one of the most widely applied techniques in biomedical research. The ease with which the technique permits specific mRNA to be detected and quantified has been a major asset in the molecular investigation of disease pathogenesis. Diseaserelated imbalances in the expression of specific mRNAs can be sensitively and quantitatively determined by RT-PCR. RT-PCR also offers many opportunities in diagnostics, allowing sensitive detection of RNA viruses such as Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV).
Mitochondrial DNA (mtDNA) typing is increasingly used for the forensic identification of human remains. This is especially true when only limited quantities of sample are present, such as when the sample has undergone extensive degradation and nucleartyping methods are ineffectual One characteristic of mtDNA responsible for the increasing reliance is the high copy number of mtDNA per cell, with
RT-PCR is an integral component of many methodologies that are essential to biomedical research, including in situ localization of mRNA, antibody engineering, and cDNA cloning. The greatest advantage of RT-PCR in the analysis of mRNA is its extraordinary sensitivity. Using nested RT-PCR, mRNA can essentially be detected at the level of single copies. The capability
PCR has become an important diagnostic as well as research tool in molecular biology, clinical chemistry, and pathology. With the invention of IS-PCR, the amplification power of solution-phase PCR with no limitations in the amount of template was hoped to be transferred to the in situ techniques.
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for such sensitive detection and analysis of mRNA is an enormous asset in many research areas, such as developmental biology. Another area that requires analysis of small local populations of cells is brain research. Understanding the function of the brain is one of the greatest challenges in biology today. Research in brain biology will benefit significantly from RT-PCR; the expression of low-abundance mRNAs can now be measured in small tissue samples from specific areas of the brain.
Identification and Differentiation of Brucella abortus Field and Vaccine Strains by BaSS-PCR Brucellosis is a bacterial disease affecting livestock worldwide. Only one assay, the AMOS assay (named for the species it identifies: B. abortus, B. melitensis, B. ovis, and B. suis), has been developed to identify and differentiate the major Brucella species and also to differentiate the B. abortus vaccine strains from field isolates. The AMOS assay is a singletube multiplex PCR assay designed to amplify up to three independent targets differing in size. Identification is based on the pattern of DNA products amplified from specific DNA targets located within the unknown isolate’s genome.
PCR Technology and Applications to Zoonotic Food-Borne Bacterial Pathogens PCR testing offers the possibility to improve detection and characterization of pathogenic bacteria, since one can target species-specific DNA regions and specific traits of pathogenicity, especially genes coding for toxins, virulence factors, or major antigens. The PCR technique has several advantages over May-June 2010 Medical Equipment & Automation
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Forensic Technology classical bacteriology with respect to detection limit, speed, and potential for automation. The latter capability is indeed necessary for application of the test in extensive screening programs. Currently, probes and PCR methods are available for many important food-borne pathogens, such as Salmonella, enterohemorrhagic E. coli, Yersinia enterocolitica, Campylobacter spp., and Listeria monocytogenes.
Long PCR Amplification of Large Fragments of Viral Genomes Long PCR has also been used to amplify the complete mitochondrial genome and large genomic fragments for the determination of deletions in genetic diseases or gene fusions in cancer. Other applications in microbiology include bacterial typing and amplification of Plasmodium falciparum DNA. Long PCR has been applied with great success to viral genomes. For example, amplification of full-length, near full-length, or large fragments of the genome of many DNA viruses has been achieved, such as for the hepatitis B virus (HBV), the human papilloma virus, SV-40, varicellazoster virus , the proviruses of the simian foamy virus of chimpanzees (SFVcpz), human T-cell leukemia virus 1 (HTLV-1), and human immunodefciency virus type 1 (HIV1) and type 2 (HIV-2). Long RT-PCR for RNA viruses has also been successful. Large fragments of the viral genome have been amplified for the tick-borne encephalitis virus, the Norwalklike viruses , the hepatitis C virus (HCV), and the bovine torovirus . The near full-length genome was amplified for HCV and the fulllength genome for the potato virus
Y, hepatitis A virus (HAV), hepatitis E virus , poliovirus , and coxsackie B2 virus.
Specificity and Performance of Diagnostic PCR Assays The undisputed success of detection assays based on the polymerase chain reaction (PCR) has been largely due to its rapidity in comparison to many conventional diagnostic methods. For instance, detection and identification of mycobacteria, chlamydiae, mycoplasmas, brucellae, and other slow-growing bacteria can be accelerated from several days to a single working day when clinical samples are directly examined. Other microbial agents that are difficult to propagate outside their natural host often remain undetected by techniques relying on cultural enrichment, thus rendering PCR the only viable alternative to demonstrate their presence. Additionally, there is the enormous potential of DNA amplification assays with regard to sensitivity and specificity. Toxoplasma gondii is an important intracellular protozoan
Prof. Mohammad Suhail
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that is the causative agent of toxoplasmosis in humans and animals. It is responsible for abortion and congenital defects in humans and is an important cause of abortion in domestic livestock, especially sheep, goats, & pigs. Direct detection of T. gondii parasites by amplification of DNA using polymerase chain reaction (PCR) has, therefore, become a valuable asset. Independent studies show that B1 locus is the best target identified so far for the routine & sensitive detection of T. gondii in human and animal tissue.
Sex Determination by PCR Analysis of the X-Y Amelogenin Gene Among known X-Y homologous genes, the amelogenin gene is the most suitable for the sex test by PCR: Following a single PCR with one pair of primers, X- and Y-specific products with different sizes are simultaneously detected because of difference in the lengths of corresponding introns. However, the sensitivity of amplification of this single-copy gene is relatively low. Then downsizing of PCR products with the use of a different set of primers and nested PCR technique has been applied to improve the sensitivity.
Prof. Mohammad Suhail, M.Sc. M.Phil. PhD is Hon. Director, City Nursing & Maternity Home Research Center, Minhajpur, Allahabad has established Department of Proteogene Life Sciences to work as its Director. He served at University of Allahabad as Reader & Head and Professor & Head, Department of Biochemistry. He has been the visiting Scientist under Teachersâ€™ Exchange Program by the Italian Education Ministry. His specialization is in Clinical Biochemistry & Molecular Biology. He has completed three Research Projects funded by Indian Council of Medical Research, UGC, New Delhi & Council of Science and Technology, U.P. Several PhD, Master of Surgery, Doctor of Medicine degrees were awarded under his guidance from University of Allahabad & its affiliated Medical College. Has chaired the 51st. Annual Meeting of the Society of Biological Chemists (India). He is the member of New York Academy of Sciences, and Life Member of National Academy of Sciences, India. He has served as expert in various academic & administrative boards and is the Reviewer of National & International Journals.
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Suhail, M. Medical Equipment & Automatiion, 2010;3 (4) p. 28-37. Director, A. Institute of Clinical Research, Civil Lines, Allahabad-211 001...