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This article explores the roles of DNA Pol I and Pol III in DNA replication and sequencing, including site-directed mutagenesis and high-throughput sequencing methods. Learn about the structure and functions of these polymerases and their importance in molecular biology.
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Anion column: DEAE-sephadex Cation column: phosphocellulose
Columns filled with porous resins. Void volume: volume of buffer surrounding the beads
Source: Chapter 20: Important feature of pol I can be cleaved by mild proteolytic treatment into two polypeptides: a large fragment (the Klenow fragment), which has polymerase and proofreading (3’→5’ exonuclease) activities; a small fragment with 5’→3’ exonuclease activity. Klenow fragment used in molecular biology when DNA synthesis required and destruction of one of the parental DNA strands, or primer Klenow fragment used to perform DNA end-filling and can used to sequence DNA Crystal structure of the Klenow fragment in 1987,------ first look at fine structure of a DNA-synthesizing machine.
Revise: DNA replication major stages and role od DNA Pol I and DNA pol III Self Reading the major DNA poIII Subunits (Chapter 21,Weaver) Source Chapter 21, Weaver The Pol III core does not function processively by itself, can replicate only a short stretch of DNA before falling template. By contrast,core plus Beta-subunit can replicate DNA processively at a rate approaching 1000 nt/sec. Beta-subunit forms a dimer :ring-shaped. This ring fits around a DNA template and interacts with the Alpha-subunit of core Experimet
What is Clamp? What is Clamp Loader?? Chapter 21: Weaver
High-throughput sequencing allows very rapid sequencing of genomes----- if genome of one member of species already sequenced. In pyrosequencing, nucleotides added one by one, and incorporation of a nucleotide detected by release of pyrophosphate---- chain of reactions to a fl ash of light. Another method ,developed by Illumina company, uses short pieces of DNA amplified in tiny, closely spaced patches on a support surface. These DNA pieces sequenced by adding fluorescent, chain-terminating nucleotides,color of whose fluorescence reveals identity. colors visualized with a microscope fitted with CCD camera. After each round of DNA elongation, fluorescent and chain-terminating groups removed and process repeated to obtain whole fragment’s sequence.
Protein Engineering with Cloned Genes: Site-Directed Mutagenesis Using cloned genes, introduce changes , thus altering amino acid sequences of protein products. Mutagenized DNA can be made with double-stranded DNA, two complementary mutagenic primers, and PCR. Simply Digesting PCR product with DpnI removes almost all of the wild-type DNA, so cells can be transformed primarily with mutagenized DNA.
Protein Engineering with Cloned Genes: Site-Directed Mutagenesis
Measure the conc. Of transcript, same labeled primer uesd for Sequencing-( used as marker for the extended labeled primer. The 5’ end-first base of transcript can be predicted
Run-off transcription: checking the efficiency and accuracy of in vitro transcription. A gene truncated in the middle and transcribed in vitro in presence of labeled nucleotides. RNA polymerase runs off end and releases an incomplete transcript. Size of this run-off transcript locates transcription start site, and amount of this transcript reflects the efficiency of transcription. In G-less cassette transcription, a promoter fused to dsDNA cassette lacking G’s in nontemplate strand, then construct transcribed in vitro in absence of GTP. Transcription aborts at end of cassette, yielding a predictable size band on gel electrophoresis.
