1887

Chapter 18 : DNA Sequencing

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $30.00

Preview this chapter:
Zoom in
Zoomout

DNA Sequencing, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555816100/9781555814717_Chap18-1.gif /docserver/preview/fulltext/10.1128/9781555816100/9781555814717_Chap18-2.gif

Abstract:

In this lesson, paper-and-paper clip simulations are used to illustrate the most common methods of DNA sequencing. Two approaches are shown, both based on the same biochemistry. The difference is that one employs a heat-resistant DNA polymerase in a modification of the polymerase chain reaction (PCR) (“cycle sequencing”), while the other uses regular DNA polymerase in a single round of reactions (“original approach”). The authors have added the cycle-sequencing simulation because it is currently the most widely employed sequencing method. The methods most commonly used for sequencing DNA on a small scale employ compounds called chain terminators, chemicals that specifically stop the elongation of a new DNA strand by DNA polymerase. Several chain terminators are also used as antiviral drugs. The reading at the end of this chapter explains how terminators work to fight human immunodeficiency virus and herpesvirus. Cycle sequencing uses the dideoxy method of sequence determination. Here, the DNA polymerase is the polymerase used in PCR, and the sequencing reactions are based on thermal cycles, as is PCR. A major difference between cycle sequencing and PCR is that only one primer is present in cycle sequencing instead of the pair used for DNA amplification. In the 1980s and early 1990s, DNA sequencing was usually performed by individual researchers in their own laboratories.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing, p 280-292. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch18

Key Concept Ranking

DNA Synthesis
0.44629985
0.44629985
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

Figures

Image of Untitled
Untitled

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing, p 280-292. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch18
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 18.5
Figure 18.5

Reaction products from the C reaction. The four shorter products were produced when a student drew a stop paper clip at the respective G in the template sequence. The long product resulted when a student happened not to draw a stop paper clip at any of the template G's.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing, p 280-292. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch18
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 18.6
Figure 18.6

The C reaction products have been “loaded” and “separated” on the “gel.” Their lengths are P 3, P 6, P 11, P 14, and P 16 (refer to Figure 18.5 ).

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing, p 280-292. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch18
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 18.7
Figure 18.7

The completed sequencing gel. The sequence is read from the bottom up: TTCGACGTAACTGCG. This is a perfect complement to the template sequence.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing, p 280-292. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch18
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 18.1
Figure 18.1

Normal deoxynucleoside shown with chain terminators. All are incorporated into DNA from their triphosphate forms.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing, p 280-292. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch18
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 18.2
Figure 18.2

DNA replication. Base pairing between an incoming deoxynucleoside triphosphate and the template strand of DNA guides the formation of a new complementary strand.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing, p 280-292. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch18
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 18.3
Figure 18.3

A student is loading a sequencing gel. A sequencing gel is what is called a vertical gel—the DNA runs from top to bottom. It is tall and quite thin: less than 1 millimeter thick. Sequencing gels are made with the substance acrylamide instead of agarose. Acrylamide forms a much tighter mesh than agarose, enabling the separation of DNA molecules that are only 1 base pair different in length.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing, p 280-292. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch18
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 18.4
Figure 18.4

Autoradiogram of a sequencing gel. The scientist who did this sequencing procedure was screening several plasmids from transformants to see if she got a clone she was trying to construct. She did. In fact, every one of the plasmids had the sequence she wanted.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing, p 280-292. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch18
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555816100.chap18a
1. Bartlett, J.,, and R. Moore. 1998. Improving HIV therapy. Scientific American279:84.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error