Reverse Transcriptase Polmerase Chain, Reaction is an Essential Tool in Molecular Biology

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

(PCR), which isolates small fragments of DNA that have a high degree of variability from

PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. Steps of the PCR cycle are shown in below figure.

There were several steps used to acquire the colony necessary for the PCR. First a student forearm was swabbed using a cotton swab, the cells were then placed in an agar plate. DNA was then extracted from the cultured bacteria by using a technique to lyse the cells and solubilize the DNA, then enzymes were used to remove contaminating proteins. The DNA extraction consisted of a lysis buffer that contained high concentrations of salt for denaturing. Binding with the use of ethanol and a washing step to purify the DNA. The final step for the DNA extraction was elution where the pure DNA was release. Proceeding the extraction of DNA the results of the 16s gene amplification were examined through gel electrophoresis it was analyzed by estimating the size of the PCR bands with marker bands. After measuring the success of the extraction, a technique called TA cloning was started. Cloning of PCR products was done by using partially purified amplified products with

While we had performed sub cellular subscription function before the function of RNA isolation in 15 cell lines to deeply interrogate the human transcription. Now for the K562 lines we had perform the additional nuclear sub fractionation into the chromatin, nucleolus and the nucleoli. The RNA from each of these sub compartment were prepare in the form of replica and were separated from each other on the basis of the length into >200 nucleotides long and <200 nucleotides short. The part consisting of the long RNA were again been further fractionated into polyadenylated and in the form of non polyadenylated transcripts. There was a use of various number of complementary technologies were been use to characterize these fractions of RNA to their sequential order was made. Sequence rewards were been mapped and were been proceed through the use of a variety of tools also called as the software tools. We have been used to map the data to assemble those and quantify De novo elements. For the process of reproducibility the elements and the quantification were been further assessed between replicates and non parametric version of the irreproducible detection rate for the statistical test. The most part of the analysis with at least 90% irreproducible. Then the data which are been

This was completed with over 65 tissue samples from various locations. The analysis was performed with quantitative PCR, using fluorescent signals.

PCR, also known as polymerase chain reaction is used

Blood samples were obtained from all family members. Genomic DNA was extracted from 100-μl samples of peripheral blood mononuclear cells incubated with proteinase K in lysis buffer, followed by phenol/chloroform extraction and ethanol precipitation. Finally, the pellet was dissolved in 30 μl sterile water and stored at -20°C until further tested. To determine mutation, specific primer sets within the APC gene were designed (primer sequences upon request). Two microliters of DNA were used to set up 25-μL PCR reactions by using the Perfect Taq plus MasterMix (5 prime GmbH, Hamburg, Germany). The amplification reaction was carried out under the following conditions; pre-incubation at 95°C for 3 min, followed by 35 cycles comprising denaturation at 95°C for 30s, primer annealing at 55°C for 30s, and extension at 72°C for 1min, and concluded by a final extension at 72°C for 5 min. Upon electrophoresis in 2.0% agarose gel stained with EtBr, the amplification products were visualized under UV

A fragment of DNA was then amplified using Polymerase Chain Reaction (PCR) with a forward primer and a reverse primer. PCR is a technique used to make as many copies as possible of a specific section of the DNA extracted. The five necessities for PCR to be successful is the DNA template that one wants to copy, a buffer, nucleotides (ATCG), polymerase, and primers. PCR occurs in 3 stages: (1) Denaturing, (2) Annealing, and (3) Extending. During the Denaturing stage, the stand containing the DNA template (in this case the fetus’) is heated to separate the double stranded DNA. Next during the Annealing stage, the stand is then cooled to allow the primers to attach to a specific location on the single stranded DNA template (primers are usually 20 to 30 bases long). Lastly during the Extending stage, the stand’s temperature is increased to permit the making of new DNA by a specific DNA polymerase. The result is multiple new strands of DNA that has been successfully copied (What is PCR, WEB), followed by electrophoresis in 1% agarose gel. Electrophoresis is a technique commonly used to separate DNA, RNA, and proteins according to their sizes. The result was then purified with a PCR purification kit (removing any remaining DNTPs, or nucleotides that could result in contamination). The products were

The initial Sanger sequencing method has been administered to several significant improvements and developed remarkably over three decades. Even though cloning of DNA fragments into plasmid vector was required in Sanger sequencing, the application of polymerase chain reaction (PCR) (Saiki et al., 1988) for the amplification of specific DNA fragment in vitro has extensively

Being able to determine the amount of DNA present in an organism has been successfully done using qPCR. In this experiment, qPCR was used to identify how much fungi could infect mutant and wildtype lines of A. thaliana. Also, qPCR was used to amplify the 16s rRNA ITS (internal transcribed spacer) of an unknown fungal pathogen. Other ways qPCR has been used in recent years include the use of qPCR to quantify copy number variants in the HER-2 gene which is a proto-oncogene. This method was particularly employed in the formalin-fixed-parafilm-embedded tissues - due to the fact that their DNA is usually broken into smaller pieces – to gain accurate results. This study concluded that qPCR produced similar results with already existing

Polymerase α is the first mammalian polymerase detected. It is a member of the B-family of replicative DNA polymerases. Its primary role is to further extend the RNA primer by addition of deoxy-nucleotides (dNTPs) into a total of 20-25 nucleotides of RNA-DNA primer. Generally, replicative DNA polymerases, polymerize dNTPs by catalyzing nucleophilic attack of the 3’-hydroxyl group of the primer onto the α-phosphate of an incoming dNTP aligned to the template strand. The accuracy of the polymerases is achieved by a confirmation of the correct base pairing of the incoming dNTP and the template strand according to Watson-Crick base-pairing. The accuracy of the primases is further enhanced (100 fold) by their exonuclease activity where in the event of a mismatch the growing strand is pushed back to the exonuclease active and the mismatched nucleotide is excised.

Further, to measure the transcript levels of foxh1 in X. tropicalis tissue fragments (early gastrulae stage) and transcription levels of the members of PouV family, the quantitative RT-PCR was used. While this method is known as a sensitive and powerful tool for analyzing of RNA samples and it has a remarkable potential for quantitative applications, its technical features require a complete knowledge of the system. Experimental variations in individual RT and PCR efficiencies should be corrected in the quantitative RT-PCR. Moreover, the expression of one or multiple housekeeping genes is essential as a reference for the analysis of testing genes’ expression levels. One of the other strong points of this research is, in fact, having appropriate control genes in the study. For example in the associated results for Figure 1-A, the transcription level of the ribosomal protein L1 (rpl1) i.e. the control gene is clearly provided.

RT PCR is one of the most useful methods used within investigated tissue to compare all transcripts accurately. To apply it accurately it has some challenges as it critically depends on the various reference materials and correct use of calibration. PCR diagnostics application to tissues is often limited by the lack of quality template due to inefficient RNA extraction methodologies (Sellars et al., 2007). Ecologically influenced or disease phenotypes and the cellular expression patterns relationship addressed with the use of gene expression profile. Enhanced information on biology of plant/microbe interactions depends on PCR–based detection technologies as utilizing species specific primers are proving indispensable as research tools (S.A. Deepak et al., 2007). Scientists interested in detecting dynamics of gene expression use RT PCR as their first choice because of high sensitivity and specificity than standard PCR which gradually proving to be insufficient for living systems (Deutsch, 2011).

2004). One- tube single step RT-PCR assay was carried out in 50 µL reaction mixture using QIAGEN OneStep RT- PCR kit (Qiagen,Ca). RT-PCR was carried out using a thermal cycler (T100 thermal cycler, Bio-Rad) to amplify cDNA fragments using the following conditions: RT at 50 °C for 30 min for reverse transcription followed by initial PCR activation for 15 min at 95 °C; 40 cycles of PCR, with each cycle at 94 °C for 60 s, 52 °C (while using PMTV2F and PMTV3 R)or 57°C ( while using C819 and H360 ) for 60 s, and 72 °C for 60 s; and a final extension at 70°C for 10 min. RT-PCR assay products were resolved by standard electrophoresis technique in pre- stained 1% agarose gels and the DNA bands were visualized under a UV-transilluminator. PCR products were cleaned by QIAquick PCR purification Kit (Qiagen) and subjected to Sanger sequencing (Retrogen, Inc. San Diego, and CA). The sensitivity of RT-PCR technique for detection of PMTV from soil extracted RNA was determined by using total RNA extracted from series of spiked soils.