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Section 9 : Advanced Techniques

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Abstract:

DNA sequence analysis is important for understanding gene structure, regulation, and function. Two methods for determining DNA sequence have been developed: the dideoxy chain termination method and the chemical cleavage method. This section first presents the basic differences between these two methods, and discusses polymerase chain reaction (PCR) can be used in DNA sequencing. The entire genome sequences of such organisms as the eukaryotes (a roundworm) and (a yeast), the prokaryotes , , and , and many viruses have been determined. Begun in the 1980s, the Human Genome Project had the goal of sequencing the entire human genome by 2005. A DNA library contains all the genetic material of a single species, broken into small fragments and cloned into a recombinant vector. DNA libraries may be constructed using genomic DNA (gDNA) or DNA complementary to RNA (cDNA), and therefore lacking intron sequences. Next, the section describes how gDNA and cDNA libraries are made, discusses how DNA libraries may be used, and explains how a gene is selected from a DNA library using filter hybridization. DNA libraries may be used to isolate specific gene sequences of interest or may be used to sequence DNA fragments whose function is not yet known. Screening DNA libraries for a specific insert may be performed by DNA hybridization or by examining clones for protein expression.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Advanced Techniques, p 251-269. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch9
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Figures

Image of Figure 28.1
Figure 28.1

(A) A dideoxynucleotide lacks a 3′-hydroxyl group. (B) Nucleoside triphosphates contain a 3′-hydroxyl. (Reprinted from B. R. Glick and J . J . Pasternak, , 2nd ed. ASM Press, Washington, D.C., 1998, with permission.)

Citation: Scheppler J, Cassin P, Gambier R. 2000. Advanced Techniques, p 251-269. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch9
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Image of Figure 28.2
Figure 28.2

Dideoxynucleotides terminate DNA synthesis because a free 3′-hydroxyl group is not available to chemically bond to additional nucleoside triphosphates. (Reprinted from B. R. Click and J. J. Pasternak, , 2nd ed. ASM Press, Washington, D.C., 1998, with permission.)

Citation: Scheppler J, Cassin P, Gambier R. 2000. Advanced Techniques, p 251-269. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch9
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Image of Figure 28.3
Figure 28.3

Schematic diagram of a silver- stained dideoxy sequencing gel. Four reactions were carried out, each with a different chain terminator (ddATP, ddCTP, ddGTP, or ddTTP). Bands were resolved by acrylamide gel electrophoresis and stained. Sequence is read from the “bottom” (short fragments) up. (Reprinted from B. R. Click and ). J. Pasternak, , 2nd ed. ASM Press, Washington, D.C., 1998, with permission.)

Citation: Scheppler J, Cassin P, Gambier R. 2000. Advanced Techniques, p 251-269. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch9
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Citation: Scheppler J, Cassin P, Gambier R. 2000. Advanced Techniques, p 251-269. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch9
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Citation: Scheppler J, Cassin P, Gambier R. 2000. Advanced Techniques, p 251-269. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch9
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Figure 28.4

Assembly of gel apparatus for the DNA acrylamide gel. (Reprinted from Bio-Rad Laboratories, Hercules, Calif., with permission.)

Citation: Scheppler J, Cassin P, Gambier R. 2000. Advanced Techniques, p 251-269. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch9
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Citation: Scheppler J, Cassin P, Gambier R. 2000. Advanced Techniques, p 251-269. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch9
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Figure 29.1

Cosmid pWE15.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Advanced Techniques, p 251-269. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch9
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References

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Tables

Generic image for table
Table 29.1

Average vector size and maximum size of DNA inserts accepted

Citation: Scheppler J, Cassin P, Gambier R. 2000. Advanced Techniques, p 251-269. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch9

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