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Section 2 : The Techniques

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Abstract:

This section talks about restriction enzyme analysis of lambda DNA, miniprep of plasmid DNA, bacterial transformation, amplification of human DNA by the polymerase chain reaction (PCR), quantitation of DNA by spectrophotometry, and quantitation of DNA by gel electrophoresis. PCR is a very powerful technique for making billions of copies of small segments of DNA. It can be performed successfully with minute quantities of DNA. It can amplify DNA that has been degraded. There are several methods to determine the quantity and concentration of DNA in a sample. These include absorption spectrophotometry, fluorescence spectrophotometry, and agarose gel electrophoresis. Although fluorescence spectrophotometry is the most sensitive, detecting DNA at concentrations of 0.01 to 15 μg/ml, absorption spectrophotometry is still the standard method. Absorption spectrophotometry detects 1 to 50 μg of DNA per ml. Quantitation of DNA by agarose gel electrophoresis provides a greater degree of sensitivity for very small samples. One can determine DNA quantities by comparing an unknown DNA with a known quantity of DNA of similar size. The section also presents exercises on topics that include restriction emyme analysis of lambda DNA, miniprep of plasmid DNA, and quantitation of DNA by gel electrophoresis.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2

Key Concept Ranking

Chromosomal DNA
0.47835454
Agarose Gel Electrophoresis
0.45220563
Simian virus 40
0.44421443
0.47835454
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Figures

Image of Figure 7.1
Figure 7.1

Unmodified DNA is cleaved by the restriction enzyme RI.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 7.2
Figure 7.2

RI is unable to cleave the methylated DNA.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 7.3
Figure 7.3

Restriction enzyme sites recognized by various restriction enzymes and the resulting ends produced after digestion.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 7.4
Figure 7.4

Schematic representation of an agarose gel electrophoresis chamber. Wells are being loaded with a micropipette.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 7.5
Figure 7.5

Semilog graph paper has a linear scale on the axis and a log scale on the axis. A log scale changes exponentially by powers of 10.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 8.1
Figure 8.1

Bacterial cell containing plasmids. Plasmids are small, circular DNA that exists extrachromosomally. Plasmids can be easily isolated from other cellular components.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 8.2
Figure 8.2

Plasmid forms isolated after a miniprep.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 8.3
Figure 8.3

Diagram of an agarose gel of plasmid DNA obtained after a miniprep. (A) Supercoiled DNA travels fastest and farthest in a gel. (B) Relaxed circular plasmid DNA travels slowest in a gel. (C) Linear plasmid DNA most accurately reflects the size.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 9.1
Figure 9.1

Plasmids probably evolved early in the evolution of living organisms by this mechanism. An internal loop forms from the normal bacterial chromosome. The ends of the loop join each other and separate from the main chromosome, forming a plasmid.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 9.2
Figure 9.2

Plasmid pGFPuv, which contains the genes for CFP and beta-lactamase. MCS, multiple cloning site.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 10.1
Figure 10.1

PCR. (A) One cycle of PCR doubles the template DNA. (B) The second and subsequent PCR cycles use the newly synthesized DNA as template DNA. DNA copies are expanded exponentially.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 10.2
Figure 10.2

The tissue plasminogen activator gene region showing no insertion (top) resulting in a 100-bp amplification fragment or with a TPA-25 insertion sequence (bottom) giving a 400-bp product. Individuals may be homozygous for either form or heterozygous.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 10.3
Figure 10.3

An example of a region of DNA containing a four-nucleotide VNTR sequence. Individual 1 has seven repeats, which will result in a 28-bp-long region of DNA. Individual 2 has 15 repeats, which will result in a 60-bp-long region of DNA. Primers for PCR bind to the region of DNA on either side of the VNTR region.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 10.4
Figure 10.4

PCR of the D1S80 locus in a family. Lane A, student's sibling; lane B, student; lane C, father; lane D, mother; lane E, maternal grandmother; lane F, maternal grandfather; lane G, PCR master mix without template; lane H, pBR322 fistNl digest DNA marker.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 11.1
Figure 11.1

A simple diagram of a spectrophotometer. Light, either UV or visible spectrum, passes through a prism and an adjustable slit. This permits selection of a specific wavelength to pass through the cuvette containing the sample to be analyzed. The light passing through the sample is collected at the phototube detector, converted into electrical current, and transmitted to the meter, where results can be measured either as percent transmittance or as optical density (absorbance).

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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Image of Figure 12.1
Figure 12.1

Diagram of typical results for quantitating DNA by gel electrophoresis. Lanes 1 to 3 and 3 to 7 are unknown samples. Lane M represents a dIII digest of bacteriophage lambda (λ) DNA, a standard DNA marker.

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
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References

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1. Ausubel, F. M.,, R. Brent,, R. E. Kingston,, D. D. Moore,, J. G. Seidman,, J. A. Smith,, and K. Struhl (ed.). 1994. Current Protocols in Molecular Biology. Wiley Interscience, New York, N.Y.
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17. Prasher, D. C.,, V. K. Eckenrode,, W. W. Ward,, F. G. Prendergast,, and M. J. Cormier. 1992. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111: 229 233.
18. Batzer, M. A.,, and P. L. Deininger. 1991. A human specific subfamily of Alu sequences. Genomics 9: 481 487.
19. Batzer, M. A.,, V. A. Gudi,, J. C. Mena,, D. W. Foltz,, R. J. Herrera,, and P. L. Deininger. 1991. Amplification dynamics of human-specific (HS) Alu family members. Nucleic Acids Res. 19: 3619 3623.
20. Budowle, B.,, R. Chakraborty,, A. M. Giusti,, A. J. Eisenberg,, and R. C. Aliens. 1991. Analysis of the VNTR locus D1 S80 by the PCR followed by high resolution PAGE. Am. J. Hum. Genet. 48: 137 144.
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24. Hochmeister, M. N.,, B. Budowle,, J. Jung,, U. V. Borer,, C. T. Comey,, and R. Dirnhofer. 1991. PCR based typing of DNA extracted from cigarette butts. Int. J. Leg. Med. 104: 229 233.
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29. Sjantilla, A.,, B. Budowle,, M. Strom,, V. Johnson,, M. Lukka,, L. Peltonen,, and C. Ehnholm. 1992. PCR amplification of alleles at the Dl S80 locus: comparison of a Finnish and a North American Caucasian population sample and forensic casework evaluation. Am. J. Hum. Genet. 50: 816 825.
30. Ausubel, F. M.,, R. Brent,, R. E. Kingston,, D. D. Moore,, J. G. Seidman,, J. A. Smith,, and K. Struhl (ed.). 1994. Current Protocols in Molecular Biology. Wiley Interscience, New York, N.Y.
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Tables

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Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Table 7.1

Standard curve data

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Table 7.2

Restriction enzyme analysis: experimental data

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Table 11.1

Absorbance data for standard curve

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Table 11.2

Absorbance data for unknown samples

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. The Techniques, p 49-95. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch2

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