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at cytosine, etc., it is possible to read the sequence of the newly synthesised strand

from the autoradiogram, provided that the gel can resolve differences in length equal

to a single nucleotide (Fig. 5.38). Under ideal conditions, sequences up to about 300

bases in length can be read from one gel.

5.11.3 Direct PCR pyrosequencing

Rapid PCR sequencing has also been made possible by the use ofpyrosequencing.

This is a sequencing by synthesis whereby a PCR template is hybridised to an

oligonucleotide and incubated with DNA polymerase, ATP sulphurylase, luciferase

and apyrase. During the reaction the first of the four dNTPs are added and if incorpor-

ated release pyrophosphate (PPi). The ATP sulphurylase converts the PPito ATP which

drives the luciferase-mediated conversion of luciferin to oxyluciferin to generate

light. Apyrase degrades the resulting component dNTPs and ATP. This is followed

by another round of dNTP addition. A resulting pyrogram provides an output of the

sequence. The method provides short reads very quickly and is especially useful for

the determination of mutations or SNPs.

Fragment to be sequenced, cloned in M13 phage

3  – – – AG – – – CTGCTCGCAT – – – 5

TC – – – GA

Primer

DNA polymerase

4 dNTPs (radioactive)

ddGTP

Synthesis of complementary second strands:

5 TC – – – GACddG 3 

5 TC – – – GACGAddG 3 

5 TC – – – GACG AGCddG 3 

Denature to give single strands

Run on sequencing gel alongside products of

ddCTP, ddATP and ddTTP reactions

ddA ddC ddG ddT Read sequence of second strand

from autoradiograph

3 

A T G C G A G

5 

Fig. 5.37Sanger sequencing of DNA.

189 5.11 Nucleotide sequencing of DNA