http://biology.hunter.cuny.edu/molecularbio/Class%20Materials%20Fall%202014%20710.LC/4th%20PF%20Lecture/Cloning%20from%20gt10%20library.pdf

Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin

Histone deacetylase is a metallo-enzyme with zinc at the active site. Compounds having a zinc-binding moiety, such as, for example, a hydroxamic acid group or a carboxylic acid group, can inhibit histone deacetylase. Histone deacetylase can repress gene expression, including expression of genes related to tumor suppression. Accordingly, inhibition of histone deacetylase can provide an alternate route for treating cancer, hematological disorders, e.g., hemoglobinopathies, and genetic related metabolic disorders, e.g., cystic fibrosis and adrenoleukodystrophy.

Histone deacetylase inhibitors

TOTAL SYNTHESIS OF MUTANTS . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 Solid Phase Chemical Synthesis of Oligodeoxyribonucleotides .. . ..... 424 Assembly of Oligodeoxyribonucleotides into Double-Strand DNA 427 Mutation by Partial Gene Synthesis (Cassette Mutagenesis) 428 OLIGODEOXYRIBONUCLEOTIDE HETERODUPLEX MUTAGENESIS . . . . . . . . . . . . . . . 428 Oligonucleotide Mutagenesis Using Single-Strand Vectors 431 Oligonucleotide Mutagenesis Using Double-Strand Vectors ........ 433 Plasmids with a Filamentous Phage Origin of Replication 435 Selection and Enrichment of Mutants 435 Screening for Mutants 437 TARGETED RANDOM POINT MUTATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438 Chemical Reagents that Change Base Pairing Specificity 439 Chemical Reagents that Eliminate Base Pairing. . . . ..... . 440 Enzymatic Incorporation of Nucleotide Analogs that Induce Mispairing ... 441 Enzymatic Misincorporation of Nucleotides.. 441 Random Point Mutations Using Long Synthetic Oligonucleotides with a Low Level of Misincorporuted Nucleotides ..... .... . ..... 443 Screening and Selection for Mutations Produced by Random Point Mutagenesis..... 444 DELETION AND INSERTION MUTAGENESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444 Deletion Mutagenesis .... .... . . 444 Linker Insertion Mutagenesis . . . . 445 IN VITRO CONSTRUCTIONS THAT TARGET IN VIVO MUTAGENESIS . . .. . . . . . . . . 446 ALLELE REPLACEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446 CHARACTERIZATION OF MUTANTS 447 In Situ Characterization of Mutants .. .... . .. . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . .... . . 447 Recovery of Genetic Functions Prior to Characterization 448 Reversion by Site-Specific Mutagenesis 448

In vitro mutagenesis

A deficiency in the plasma protease inhibitor α1-antitrypsin can cause chronic obstructive emphysema or infantile liver cirrhosis. This deficiency results from a single amino acid substitution created by a G to A transition in the gene for α1-antitrypsin. Chemically synthesized specific oligonucleotide probes (19-mer) have been used to develop a sensitive and direct test for the presence or absence of the mutant gene in any individual, which can be used for prenatal diagnosis of the deficiency syndrome.

alpha 1-antitrypsin deficiency detection by direct analysis of the mutation in the gene.

A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322

A solid phase method for the simultaneous synthesis of mixed oligonucleotides using a phosphotriester approach has been developed. For this synthesis, a mixture of mono or dimeric coupling units is used, and a slight difference in the reactivity of those units is found. However, this difference does not hamper the simultaneous, mixed oligonucleotide synthesis, and the sequence analysis of a product demonstrates the existence of all desired sequences in the final mixture.

Solid phase synthesis of polynucleotides. VIII. Synthesis of mixed oligodeoxyribonucleotides by the phosphotriester solid phase method1

Preparation of the three hexadecanucleotides, dGpTpApTpCpApCpGpApGpGpCpCpCpTpT, dCpGpApCpGpApGpCpGpTpGpApCpApCpC and cTpGpCpCpGpGpCpCpApCpGpApTpGpCpG, is described by a rapid and simple solid-phase method on polyacrylamide supports. The synthesis were performed by the extension of the method described in the previous paper using di and trinucleotides of defined sequences as an incoming 3'-phosphodiester unit. Although the coupling yields to form phosphotriester bonds are slightly lower than those for the homothymidylic acid series, pure polydeoxyribonucleotides of defined sequences can be synthesized without any major difficulty.

/pdf/solid-phase-synthesis-of-polynucleotides-iii-synthesis-of-415h016nc2.pdf

Solid-phase synthesis of polynucleotides. III. Synthesis of polynucleotides with defined sequences by the block coupling phosphotriester method

Contrary to the expectation, the Merrifield polystyrene resin, 2% cross-linked by divinylbenzene, is as efficient as the polyacrylmorpholide resin for the synthesis of polydeoxyribonucleotides using a phosphotriester method. On the Merrifield resin, the tetradecamer, dTpCpGpTpCpApApCpTpGpGpCpTpT, and the hexadecamer, dCpCpApGpTpCpApCpGpApCpGpTpTpGpT, were synthesized by the phosphotriester method using di and trinucleotide blocks as coupling units.

/pdf/solid-phase-synthesis-of-polynucleotides-iv-usage-of-38mualk8g7.pdf

Solid-phase synthesis of polynucleotides. IV. Usage of polystyrene resins for the synthesis of polydeoxyribonucleotides by the phosphotriester method

Publisher Summary This chapter discusses the role of synthetic DNA in the preparation of structural genes coding for proteins. One of the key steps in the production of proteins by recombinant DNA technology is the preparation of structural genes coding for the desired proteins. There may be two approaches to this goal: (1) artificial gene synthesis and (2) a combination of cloned cDNA sequences and synthetic DNAs. The method of artificial gene synthesis for the preparation of artificial genes was developed for the synthesis of genes for tRNAs in the late 1960s. The approach of combination of cloned cDNA sequences and synthetic DNA has been developed for the preparation of a gene coding for the human growth hormone. The chapter describes an alternative approach for the synthesis of polydeoxyribonucleotides with defined sequences longer than those accessible by conventional chemical methods. Recent advances in the chemical synthesis of oligonucleotides help to synthesize single-stranded oligonucleotides of up to 40 bases in length, rapidly. It is now possible to synthesize double-stranded DNA fragments using DNA polymerase I in the presence of four deoxynucleoside triphosphosphates.

Ting Huang

Papers

Solid phase synthesis of polynucleotides. VIII. Synthesis of mixed oligodeoxyribonucleotides by the phosphotriester solid phase method1

Solid-phase synthesis of polynucleotides. III. Synthesis of polynucleotides with defined sequences by the block coupling phosphotriester method

Solid-phase synthesis of polynucleotides. IV. Usage of polystyrene resins for the synthesis of polydeoxyribonucleotides by the phosphotriester method

The role of synthetic dna in the preparation of structural genes coding for proteins