Chapter 13
Sequencing
KLAUS DEICHMANN
lntroduction
Principles of sequencing
Knowledge of the DNA primary sequence leads to a closer understanding of
the structural organization of DNA, the deduction of an amino acid sequence and allows for the analysis of ( clinically relevant) mutations. The
first sequencing methods were quite labor-intensive and time-consuming
(Wu and Taylor 1971; Robertson et al. 1973; Sanger et al. 1973; Ziff et al.
1973). However, two rapid and universally applicable methods have
emerged: Maxam and Gilbert used chemical agents producing defined fragments ofDNA (Maxam and Gilbert 1977). Thesefragmentsare madevisible
by polyacrylamide gel electrophoresis using radioactive markers. The original DNA sequence can be deduced from the length of fragments formed.
The second technique has been developed by Sanger in 1977 (Sanger et al.
1977). This method, the chain termination method, is most widely used
nowadays, and is based on enzymatic steps (Davis et al. 1986; Sambrook
et al. 1989; Ausubel et al. 1990).
The chain termination method
Usually, the DNA fragmenttobe sequenced, the insert, is cloned into a vector, a DNA molecule ofknown structure and sequence. Sequencing is performed on this construct in a single-stranded state. Single-strand DNA can
be obtained, either by denaturation of double-stranded DNA or directly by
the use of single-stranded vectors. An oligonucleotide primer, that is comKlaus Deichmann, Universitäts-Kinderklinik, Mathildenstrasse 1, Freiburg, 79106, Germany (phone +49-7612704300; fax +49-7612706372; e-mail deichman@kkl200.ukl.unifreiburg.de)
Sequencing
KLAUS DEICHMANN
lntroduction
Principles of sequencing
Knowledge of the DNA primary sequence leads to a closer understanding of
the structural organization of DNA, the deduction of an amino acid sequence and allows for the analysis of ( clinically relevant) mutations. The
first sequencing methods were quite labor-intensive and time-consuming
(Wu and Taylor 1971; Robertson et al. 1973; Sanger et al. 1973; Ziff et al.
1973). However, two rapid and universally applicable methods have
emerged: Maxam and Gilbert used chemical agents producing defined fragments ofDNA (Maxam and Gilbert 1977). Thesefragmentsare madevisible
by polyacrylamide gel electrophoresis using radioactive markers. The original DNA sequence can be deduced from the length of fragments formed.
The second technique has been developed by Sanger in 1977 (Sanger et al.
1977). This method, the chain termination method, is most widely used
nowadays, and is based on enzymatic steps (Davis et al. 1986; Sambrook
et al. 1989; Ausubel et al. 1990).
The chain termination method
Usually, the DNA fragmenttobe sequenced, the insert, is cloned into a vector, a DNA molecule ofknown structure and sequence. Sequencing is performed on this construct in a single-stranded state. Single-strand DNA can
be obtained, either by denaturation of double-stranded DNA or directly by
the use of single-stranded vectors. An oligonucleotide primer, that is comKlaus Deichmann, Universitäts-Kinderklinik, Mathildenstrasse 1, Freiburg, 79106, Germany (phone +49-7612704300; fax +49-7612706372; e-mail deichman@kkl200.ukl.unifreiburg.de)