PCR be unique to avoid amplifying other sections

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Last updated: March 18, 2019

PCR is a molecular test used to detectand copy target regions of DNA. It amplifies sections of DNA by mimickingnatural DNA replication using DNA polymerase(1). Forward and reverse primers,complementary to the start and end of the DNA region, are used to flank thetarget sequence.

PCR is performed using thermal cyclingthrough stages of denaturation, annealing and extension. Firstly, hydrogenbonds between the double helix of the template DNA are broken, then the primersbind to the now single stranded target DNA, DNA polymerase recognises these andstarts adding complementary nucleotides to each strand. This leaves twodaughter strands of DNA for the next cycle, leading to exponentialamplification. The products are then analysed using electrophoresis orhybridisation(1).The limitations of PCR are that thetarget sequence of DNA needs to be already known so that primers can bedesigned. The sequence also needs to be unique to avoid amplifying othersections of DNA in the sample, this can also lead to wrong sections of DNAbeing amplified if the specimen is contaminated and the primers aren’t specificenough. There is a maximum length of DNA that can be synthesised which means itis less useful for diseases with trinucleotide repeats such as Huntington’s(1). 1.

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1       Applications Detecting Ig/TCR Gene RearrangementsAt the early stages of B and T celldevelopment random coupling between one of many Variable (V), diversity (D) andjoining (J) genes results in the formation of a unique V(D)J exon of theantigen binding section of immunoglobulins (Ig) and T Cell Receptors (TCR)(3). This leads to each lymphocyte havinga unique antigen receptor molecule on its membrane.  If more than one lymphocyte has the samereceptor it must be a clone, as the likelihood of these occurring separately isnegligible. In a healthy individual, the polyclonal repertoire of Ig and TCRmolecules is over 1012(3).Standardised multiplex PCR assays fornearly all Ig/TCR targets are used in suspected lymphoid proliferations(4).

These are then interpreted; peaks orbands are results of a monoclonal population as the products produced are ofthe same size and sequence. Whereas a normal polyclonal population results in asmear due to differently sized PCR products(4). BIOMED-2 standardized protocols havebeen developed so detection should be reliable across different labs(4).

Normal agarose gel to analyse PCRproducts is not high enough resolution so results in false positives. Insteadgenetic fragment analysis or heteroduplex analysis is needed(3).1.

2       Applications Detecting FLT3 MutationsFLT3 is a receptor tyrosine kinaseimportant in normal haematopoiesis, a mutation results in constitutiveactivation and is seen in acute myeloid leukaemia (AML)(5). Two common mutations are internaltandem duplications (ITDs) and a D835 mutation(6). ITDs are found in 20-30% AML and are associatedwith an adverse prognosis(5,7). FLT3 ITD mutations are associatedwith an increased risk of relapse and reduced overall survival(6). PCR is useful to detect thesemutations as they are limited to a small predictable region of the gene.

D835 mutations are found in 7% cases(5,7,6). It is a missense mutation and eliminatesan EcorV restriction enzyme site. PCR is used to amplify the section of DNAwith the FLT3 gene, the primers flank the possible mutation and another EcorVsite which is used as a control. The product is then digested with EcorV, anormal result is an 80bp product where EcorV has cut at both sites.

A mutationresults in a 120bp product, where EcorV has only cut at one site as the secondhas been eliminated by the mutation(1). 2        Quantitative PCR (QPCR)2.1       OverviewQPCR is a method developed fromtraditional PCR used to amplify and detect DNA concurrently. A fluorescentsignal is produced at the same time as product formation. This is done with afluorescent dye that intercalates with double stranded DNA or specific oligonucleotideprobes that fluoresce when they hybridise with complementary DNA. The PCRcycler monitors fluorescence produced during annealing or at the end of each PCRcycle and creates a plot of fluorescence intensity versus cycle number. Thecycle threshold is when the intensity crosses a predetermined threshold and isproportionate to the amount of starting template(1).2.

2       Applications Detecting BCR/ABLIn Chronic Myelogenous Leukaemia (CML)the Philadelphia chromosome is formed by a reciprocal translocation t(9;22) ofBCR and ABL genes(8). The BCR gene translocates to exon 2of the ABL1 gene leading to a 210 or 190 kDa fusion protein(9). This product is a constitutivelyactive tyrosine kinase protein which causes uncontrolled cell proliferation.

CML is treated by Imatinib, a selectiveinhibitor of BCR-ABL tyrosine kinase, which has led to an overall survival forimatinib treated CML of 85%(10). Not all CML cells are eradicated bythis treatment, so it has to be life long and relapse can occur. There are 31different point mutations which are detected in 35-90% resistant patients(8). Acquired resistance from thesemutations leads to reactivation of BCR-ABL tyrosine kinase. Therefore, it isimportant to monitor patients with CML.QPCR can be used for this. Levels ofthe BCR-ABL fusion protein can be detected pre-treatment and monitored overtime to see if it is changing or stable(11).

A rise in BCR-ABL of more thantwo-fold detected by QPCR is a primary indicator to test patients for BCR-ABLkinase domain mutations by sequencing(8). The level of BCR-ABL is predictive ofdisease-free survival and rising levels are predictive of relapse afterallogeneic transplantation(8).Reverse transcriptase QPCR is oftenused as mRNA is more abundant than DNA, but DNA QPCR has a higher sensitivity.FISH is able to detect rare translocations not detectable by commercial QRT-PCR. Although, specialised labs with multiplex QPCR using hundreds ofprimers can also detect these(9).

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