Recombinant DNA sequencing

FIGU RE 3.12 Using sticky ends generated by the action of a restriction endonuclease to recombine DNA sequences. 

According to the opponents, the main argument against granting a patent was the lack of novelty of the recombinant DNA sequences claimed, which included the DNA sequences that coded interferon-alpha (IFN-alpha) as an insert. This prior art referred to DNA genome libraries, which are fractioned segments (15-20 Kb) of chromosomal DNA of human fetuses, cloned as derivatives of recombinant X phages. 

Colony hybridization A technique that is used to screen bacteria for the presence of a specific recombinant DNA sequence. Colonies of the bacteria are transferred to a filter, treated to lyse the cells and denature the DNA, and then exposed to a labeled DNA probe that is complementary to part of the sequence in question. Colonies that bind the probe possess the sequence. 

Workers in the early 1970s recognized that restriction enzymes provided tools not only for DNA mapping but also for constmction of new DNA species not found in nature. A collection of recombinant DNA species consisting of many passenger sequences joined to identical vector molecules is called a hbrary. Individual recombinant DNAs are isolated from single clones of the Hbrary for detailed analysis and manipulation. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

After a desired clone is obtained and mapped with restriction enzymes, further analysis usually depends on the deterrnination of its nucleotide sequence. The nucleotide sequence of a new gene often provides clues to its function and the stmcture of the gene product. Additionally, the DNA sequence of a gene provides a guidepost for further manipulation of the sequence, for example, lea ding to the production of a recombinant protein in bacteria. 

Replication of the Recombinant DNA. In bacteria, repHcation of the recombinant DNA is provided by origin sequences, derived usually... 

Methionyl hGH. The first form of hGH to be produced through recombinant DNA technology was actually a derivative of hGH having one additional methionine residue at its N-terminus (11). Although technology has advanced to the stage where natural sequence hGH can easily be produced, as of this writing this derivative, referred to as methionyl hGH, is stiU produced commercially. 

Both catenanes and knots can bring together remote DNA sequences and may be important in transcription regulation and genetic recombination... 

Recombinant DNA techniques have provided tools for the rapid determination of DNA sequences and, by inference, the amino acid sequences of proteins from structural genes. The number of such sequences is now increasing almost exponentially, but by themselves these sequences tell little more about the biology of the system than a New York City telephone directory tells about the function and marvels of that city. 

This chapter describes the chemistry of nucleotides and the m or classes of nucleic acids. Chapter 12 presents methods for determination of nucleic acid primary structure (nucleic acid sequencing) and describes the higher orders of nucleic acid structure. Chapter 13 introduces the molecular biology of recombinant DNA the construction and uses of novel DNA molecules assembled by combining segments from other DNA molecules. 

Therefore, DNA restriction fragments having such sticky ends can be joined together to create new combinations of DNA sequence. If the fragments are derived from DNA molecules of different origin, novel recombinant forms of DNA are created. 

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