...

Sequencing of Nucleotides in DNA

by taratuta

on
Category: Documents
94

views

Report

Comments

Transcript

Sequencing of Nucleotides in DNA
Page 671
Figure 15.49 Genomic rearrangements promoted by transposons. In replicative transposition a transposable element is replicated, with one copy of it remaining at the donor site and a new copy becoming inserted within a different location. This produces two homologous sequences within the same chromosome that can subsequently enter into homologous recombination. (a) When these homologous sequences are of the same polarity, recombination can yield a deletion of DNA by a process that is superficially analogous, but certainly not similar, to the reverse reaction occurring in site­specific recombination. (b) Inversion of DNA flanked by these transposons can result when transposable elements are present in the chromosome oriented in opposite direction. Redrawn based on figure from Mathews, C. K. and Van Holde, K. E. Biochemistry. Redwood City, CA: Benjamin/Cummings, 1990.
15.6— Sequencing of Nucleotides in DNA
Restriction Maps Give the Sequence of Segments of DNA
The sequences of many genes and adjoining DNA segments have been determined for bacteria, viruses, plants, and humans. The determination of the sequence of a large DNA molecule begins by cutting the DNA into pieces of a more manageable size with appropriate restriction endonucleases. Restriction digests permit the construction of a characteristic restriction map for each DNA. One protocol depends on the generation of partial restriction digests of end­labeled DNA. Partial digests are obtained by setting the conditions so that
CLINICAL CORRELATION 15.10 DNA Amplification and Development of Drug Resistance
An important limitation in the effectiveness of chemotoxic drugs in the treatment of cancer is the development of drug resistance. Thus cancer cells become resistant to methotrexate (see p. 520), an inhibitor of dihydrofolate reductase (DHFR). Drug resistance in cultured cells results from the specific amplification of a large DNA segment that incorporates the DHFR gene but the exact mechanism by which amplification occurs is not clear. It appears likely that amplification results from recombination of identically oriented homologous sequences that flank the amplified DNA. Amplification can occur by tandem duplication of DNA that contains the DHFR gene or alternatively the DHFR­containing segment can be excised (apparently by a recombination process), producing extrachromosomal DNA (minichromosomes). The two mechanisms of DHFR gene amplification are not mutually exclusive and, in fact, some resistant cells contain both types of amplified DNA.
Gene amplification is gradually reversed in the absence of methotrexate, first with the disappearance of the extrachromosomal copies. Chromosomally amplified genes, however, persist for several generations after removal of the drug. The amplification of genes is a general phenomenon not limited to methotrexate or the development of cell resistance toward other drugs. In fact, gene amplification and the accompanying resistance extend to areas well beyond clinical medicine, as, for instance, in agriculture with the development of pesticide­resistant insects.
Page 672
CLINICAL CORRELATION 15.11 Nucleotide Sequence of the Human Genome
The purpose of the Human Genome Project is to provide a detailed map of the human genome and establish what DNA sequences determine human phenotypic characteristics and guide human development. A corollary to this goal is to identify genes responsible for human disease so that new approaches can be developed for diagnosis, prevention, and therapy.
The human genome is believed to consist of 70,000–100,000 different genes that determine the genetic characteristics of every cell in the human individual. The human genome consists of about three billion base­paired nucleotides that are assembled in the form of 23 pairs of chromosomes. The availability of restriction endonucleases and the development of effective physical mapping procedures for DNA, combined with the increasing rapidity of contemporary nucleotide sequencing methods, have provided strong impetus for the very ambitious undertaking of determining the nucleotide sequence of the entire human genome.
Extensive physical mapping has been completed. In addition, genetic mapping seeks to locate over 500 known genetic markers on the human chromosomes. Cumulatively over 150 million base pair sequences, representing parts of the chromosome sequences of both human DNA and that of other organisms, have been determined. Also, the sequences of certain continuous stretches of DNA, ranging from one million to several million base pairs in length, are being determined. Considering that the size of different human chromosomes varies from 263 million to less than 50 million base pairs, the determination to date of a total of about 150 million base pairs represents an important accomplishment. It is conservatively estimated that complete sequencing of the genome will take more than a decade and a half.
Because of the routine nature of determining the nucleotide sequences involved, many scientists have questioned the wisdom of diverting resources from perhaps more creative scientific endeavor, to the effort required to sequence the human genome. Others have pointed out that the project is fraught with technical uncertainties. Proponents point out the great potential benefits of determining the imprint that controls the genetic properties of the human cell at the highest possible level of resolution. Presently, as many as 4000 genetic diseases have been identified and many of them, namely, those inherited in Mendelian fashion, are caused by a single mutant gene. Searching for the imprint of human disease at the level of nucleotide sequences may permit understanding of all disease states at the genomic level. Determination of the complete sequence appears to be one of the prerequisites for understanding human disease at the molecular level. There is little doubt that the sequencing of the human genome will present us with many new challenges and opportunities in medicine.
Grant Cooper, N. (Ed.). The Human Genome Project. Mill Valley, CA: University Science Books, 1994.
the restriction endonuclease will not recognize all sites in every DNA molecule but will instead produce a digest that includes a collection of partial fragments. Double­
stranded DNA is end­labeled by treatment with alkaline phosphatase, which removes the phosphate residue at the 5 end, and then g­labeled with [32P]ATP and a polynucleotide kinase, which incorporates the 32P into the two 5 termini of the DNA strands. Alternately, the 32P­label can be introduced at the 3 termini by the incorporation of 32P­labeled deoxyribonucleotide triphosphates using DNA polymerase. End­labeling allows for each fragment to be identified on an electrophoresis gel. The details of this procedure are presented on page 762. Thus, with a series of different site cuts, the fragments can be mapped directly relative to the labeled end. Restriction maps are used for characterization of various DNAs and for ordering of smaller DNA fragments within a particular DNA sequence. Such ordering is essential before the nucleotide sequence of large DNA molecules can be determined.
Several methods have been developed for rapid sequencing of large poly­deoxyribonucleotides. They are impressively accurate. Digests obtained using different restriction enzymes produce segments with overlapping lengths of nucleotide sequences. The accuracy of sequencing methods are increased by sequencing the complementary strand. These procedures can also be used for sequencing of RNA molecules by prior conversion of the RNA sequence to a complementary DNA by use of reverse transcriptase. Sequences up to 500 bp can be determined in a single automated operation and stretches of 10,000 bp, which correspond to the average length of a gene, are now routinely determined. Clinical Correlation 15.11 discusses the application of these procedures for obtaining the sequence of the human genome.
Fly UP