Direct DNA Sequencing Using PCR (Also Called Ligation Mediated PCR = LMPCR),Polymerase Chain Reaction,DNA Sequencing,Denaturation

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Isolation, Sequencing and Synthesis of Genes Isolation, Sequencing and Synthesis of Genes - Introduction Isolation of Genes Isolation of Genes Coding for Specific Proteins Isolation of Genes with Known or Unknown Products having Tissue Specific Expression Isolation of Genes with Known or Unknown Products Using DNA or RNA Probes Use of Heterologus Probes Use of Synthetic Probes Isolation of Genes Coding Use of Transposable Elements Transposon Tagging T-DNA-Insertion Mutagenesis for Isolation of Plant Genes Promoter Enhancer and Gene Trap for Isolation of Genes Differential Screeing and Differential Display Technique for Isolation of Genes Subtractive Hybridizatioon for Gene Isolation Map Based Cloning for Gene Isolation Isolation of Novel Genes by Asis Data at JNU New Delhi

Home >> Biotechnology and Genomics >> Isolation, Sequencing and Synthesis of Genes >> Direct DNA Sequencing Using PCR (Also Called Ligation Mediated PCR = LMPCR)

Your Ad Here	Direct DNA Sequencing Using PCR (Also Called Ligation Mediated PCR = LMPCR) Polymerase chain reaction (PCR) has also been used for sequencing the amplified DNA product. This method of DNA sequencing is faster and more reliable and can utilize either the whole genomic DNA or cloned fragments for sequencing a particular DNA segment. The DNA sequencing using PCR involves two steps: (i) generation of sequencing templates (double stranded or single stranded) using PCR and (ii) sequencing of PCR products either with the thermolabile DNA polymerase or with the thermostable Taq DNA polymerase. Thus the DNA sequencing method using PCR eliminates the need of cloning the DNA in single stranded DNA phage vector i.e. M13.
Although double stranded DNA product of PCR can be utilized for sequencing, this may cause, difficulty due to reassociation after denaturation thus preventing the sequencing primer from annealing to its complementary sequence to allow extension. To reduce this problem, either a variant of the standard method for sequencing double stranded DNA is employed or single stranded DNA templates are produced following asymmetric PCR earlier discussed in. A number of thermolabile DNA polymerases have been used for sequencing of in vitro amplified DNA. Alternatively, Taq polymerase (thermostable enzyme) used for PCR, can also be used for sequencing reaction. In either case, Sanger's synthetic method involving incorporation of dideoxynucleotide (ddNTP) for chain termination, is used for sequencing. However, Maxam and Gilbert's chemical degradation method can also be used.
In Sanger's method, as usual, four mixtures are prepared, each using one of the four ddNTP. The sequencing primer is labelled with 32p and the mixtures with amplified DNA, Taq polymerase and appropriate buffer are incubated at 700C for 5 min. The reaction is stopped by addition of formamide stop solution in each tube and mixtures are run on polyacrylamide sequencing gel to obtain the ladders, which can be read by computer or manually. This method using PCR helped in automation of DNA sequencing.	Your Ad Here

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Sequencing of a Gene or a DNA Fragment Maxam and Gilbert's Chemical Degradation Method Sanger's Dideoxynucleotide Synthetic Method Automatic DNA Sequencers Slab Gel Versus Capillary Sequencers Slab Gel Sequencing Systems Capillary Gel Sequencing Systems Direct DNA Sequencing Using PCR Ligation Mediated PCR LMPCR DNA Sequencing Through Transcription DNA Sequencing by Hybridization Using Microarrays on DNA Chips DNA Sequencing by DE-MALDI-TOF Mass Spectrometry Synthesis of Genes Synthesis of Gene for Yeast Alanyl tRNA Synthesis of Oligonucleotides Synthesis of Three Duplex Fragments Synthesis of Gene from Three Duplex Fragments Synthesis of Gene for a True Precursor tRNA Total Synthesis of a Human Leukocyte Interferon Gene Gene Synthesis Machines Protection Chemistry and Amidites Permanent Protecting Groups Temporary Protecting Groups Synthesis of Gene Using PCR Synthesis of Genes From mRNA