PCR Sequencing Protocols

1. Front Matter

   Pages i-xi

   PDF

2. Preparation and Analysis of DNA Sequencing Gels

   • Bimal D. M. Theophilus

   Pages 1-9

3. Purification of PCR Products from Agarose Gels for Direct Sequencing

   • Frank C. Brosius III, Lawrence B. Holzman, Xinan Cao

   Pages 11-21

4. Enzymatic Fluorescence and Biotin Labeling of Primers for PCR Sequencing

   • Gabor L. Igloi

   Pages 23-28

5. Direct Sequencing of Double-Stranded PCR Products with the Sequenase Kit and [α-³⁵ S] dATP

   • Jean-Laurent Casanova

   Pages 29-34

6. Direct Sequencing by Thermal Asymmetric PCR

   • Georges-Raoul Mazars, Charles Theillet

   Pages 35-40

7. Rapid Sequencing of cDNA Clones Direct Sequencing Using Sequential Linear/Asymmetric PCR

   • Ivor J. Mason

   Pages 41-47

8. Direct Sequencing of PCR Products Using Chemiluminescent Detection

   • Bentley A. Atchison, Andrea M. Douglas

   Pages 49-55

9. Direct DNA Sequencing of PCR Products Using Magnetic Beads

   • Joakim Lundeberg, Bertil Pettersson, Mathias Uhlén

   Pages 57-66

10. Affinity Capture and Solid-Phase Sequencing of Biotinylated PCR Products

    • Anu Suomalainen, Ann-Christine Syvänen

    Pages 67-72

11. Analysis of Nucleotide Sequence Variations by Solid-Phase Minisequencing

    • Anu Suomalainen, Ann-Christine Syvänen

    Pages 73-79

12. Nonradioactive PCR Sequencing Using Digoxigenin

    • Siegfried Kösel, Christoph B. Lücking, Rupert Egensperger, Manuel B. Graeber

    Pages 81-89

13. Silver Sequencing™

    • Alison Wade-Evans

    Pages 91-100

14. Direct Sequencing of PCR Products with DNA-Binding Proteins

    • Ralph Rapley

    Pages 101-104

15. PCR Sequencing with the Aid of Detergents

    • Harald Petry, Barbara Bachmann, Wolfgang Lüke, Gerhard Hunsmann

    Pages 105-109

16. Direct Sequencing with Highly Degenerate and lnosine-Containing Primers

    • Zhiyuan Shen, Jingmei Liu, Robert L. Wells, Mortimer M. Elkind

    Pages 111-118

17. Determination of Unknown Genomic Sequences Without Cloning

    • Jean-Pierre Quivy, Peter B. Becker

    Pages 119-131

18. DNA Sequencing by the Chemical Method

    • Eran Pichersky

    Pages 133-136

19. Direct PCR Sequencing with Denaturants (Formamide)

    • Wei Zhang, Albert B.

    Pages 137-147

20. Efficient PCR Production of Single-Stranded DNA Sequencing Templates

    • Bernhard Kaltenboeck, Konstantin G. Kousoulas

    Pages 149-153

Advances in bioscience research usually arise as a result of the continu­ ing refinement of existing technologies. However, there are a number of occa­ sions v^rhere newly developed methodologies have a profound effect on nearly all areas of research. Frequently these are techniques that are elegantly simple in concept and require minimal technical manipulation. Two of these revolu­ tionary techniques are the focus ofPCR Sequencing Protocols. The first such technique is enzymatic chain termination sequencing developed by Sanger and his co-workers in Cambridge and reported in 1977. This essentially brought the possibility of deriving nucleotide sequence information in a very short time scale and has been widely accepted in many laboratories as a routine molecular biological research tool. Furthermore, it has not only led to the sequencing of many genes and gene fragments, but has also allowed the tech­ nical means of sequencing the human genome. The second technique that has found widespread acceptance in basic applied research and many routine applications is the polymerase chain reac­ tion. This technique, first reported in 1985 by MuUis and his colleagues, pro­ vides the means to amplify nucleic acid sequence, which immediately proved invaluable in nearly all fields of biological laboratory research. Here, as with enzymatic DNA sequencing, is a very simple concept that relies on minimal information to prepare short oligonucleotide primers that direct the synthesis of a specified fi-agment o f DNA in the presence of a thermostable DNA polymerase.

...this is a good collection of well presented PCR protocols that should find its way into many laboratories...if you are planning on performing a large amount of PCR sequencing, or have been having problems sequencing PCR products, this book has a procedure or advice that will help. -FEBS Letters

"The book is a must for investigators in molecular biology and involved with sequencing and setting up PCR protocols....I highly recommend this most comprehensive volume for all molecular biologists."- SMG Quarterly

• School of Natural Sciences, Coventry University, Coventry, UK

  Ralph Rapley

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