Oligonucleotide synthesis

protection Manoharan et al. (1999) developed 2(cyanoethoxycarbonyloxy)succinimide S.27a a stable, crystalline, and convenient reagent for the protection of pendant akylamines in oligonucleotides (Figure X.X.X). The 2-cyanoethyl (ce) group is the most common phosphate protecting group in oligonucleotides; however, ce and corresponding 2-(cyanoethoxycarbonyl) group were not utilized for the nucleobase protection until 2000. Merk et al. (2000) developed 2-(cyanoethoxy)carbonyl (ceoc)…

alkaline conditions. The 5,6 double bond of pyrimidine nucleosides also reacts with halogens and halohydrins to give the corresponding addition products (Shabarova and Bogdanov, 1994). Selected examples of the side reactions that occur during oligonucleotide synthesis are given below. Tri-O-acetyluridine (S.1) reacts with MSNT to produce the triazolo derivative S.2 (Figure 2.1.2; Reese and Ubasawa, 1980). During deprotection with ammonium hydroxide, S.2 gives cytidine (S.3). Interestingly,…

temperature regulation is essential to optimize the results. The length of the DNA region to be copied depends on the cycle repeats (25 - 40) and time (2 - 4 hours). The new DNA made in one round can serve as a template in the next round of DNA synthesis. The number of DNA molecules double in each cycle due to the multiple copies of primers and Taq polymerase. The copy number is the number of amplified DNA copies produced in the PCR. It is calculated by multiplying the Avogadro number by the…

regarding PCR. PCR was developed by a combination of several techniques already in existence; the synthesis of oligonucleotides and their utilization to synthesize new copies of DNA that were specific using DNA polymerases. Mullis specifically used two oligonucleotides that were each complementary to an opposite strand of the DNA (Bartlett, 2003). This allowed the region in between the oligonucleotides to become amplified and Mullis did this in repetition, allowing the product formed from DNA…

importance in the hot spot. Residues are substituted randomly using techniques such as overlap- extension PCR (oe-PCR) and QquickC change mutagenesis PCR (QC-PCR). The former is popular for introducing multiple mutations in the gene. Degenerate oligonucleotides are used as primers in PCR to amplify fragments of the gene. Each amplified fragment contains a region of homology with the appropriate adjacent gene fragment. Fragments are mixed, similarly to DNA shuffling, in the absence of primer…

template undergoes exponential amplification products obtained for each cycle using the following cycles matrix. Replication involves three steps: (1) denaturation of double-stranded DNA into single-stranded template; (2) hybridization of the oligonucleotides…

incorporation of nucleotide to new DNA strand during sequencing run. Four main steps summarises the entire sequencing technology, that have described in 454 sequencing website, (i) DNA library preparation, (ii) Emulsion PCR, (iii) Sequencing-by-synthesis…

Q1) What secondary structures make up the large C-terminal domain of the Klenow fragment? Answer: The large C-terminal domain contains a six-stranded antiparallel β-sheet at the bottom of the cleft region and contains three α-helices on one side and four α-helices on the other side of the cleft region. Q2) Describe the three-dimensional structure of the large C-terminal domain of the Klenow fragment? Answer: The large C-terminal domain contains a significantly deep cleft, with a considerable…

T. Heinze et. al. reported the synthesis of new 6-deoxy-6-pheylethylamino cellulose derivatives with DSamino 0.4-0.6 by nucleophilic displacement of cellulose tosylates (DSTos 0.74 and 1.29) with a racemic mixture of 1-phenylethylamine under homogeneous conditions, in DMF/H2O82, scheme 30. The optical properties of these deoxyamino cellulose derivatives was studied. A regioselective synthesis of 6-amino-6-deoxycellulose, 6-N-sulfonated, and 6-N-carboxymethylated deoxycellulose were…

3.3.1 POLYMERASE CHAIN REACTION The Polymerase Chain Reaction (PCR) is the most common DNA amplification method in molecular biology, it was invented by Kary Mullis while working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. His invention won him a Nobel Prize in Chemistry in 1993. PCR has revolutionized the field of molecular biology as it has enabled researchers to perform experiments easily that previously had been unthinkable. Before the mid-1980s…