Primer walking cycle sequencing using presynthesized primers con

Details Primer walking cycle sequencing using presynthesized primers con Primer walking cycle sequencing using presynthesized primers con

1998-03-23

2000-03-28

Horlick, Kenneth R.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 912, 435810, 536 2433, C12P 1934, C07H 2104

Patent

active

060430591

DESCRIPTION:

BRIEF SUMMARY
INTRODUCTION

In primer walking sequencing, a primer/template complex is extended with a polymerase and chain terminated to generate a nested set of fragments whence the sequence is read after electrophoresis and detection (radioactive or fluorescent). A second primer is then synthesised using the sequence information near to the end of the sequence obtained from the first primer. This second (`walking`) primer is then used for sequencing the same template. Primer walking sequencing is more efficient in terms of generating less redundant sequence information than the alternative `shotgun` approach.


DISADVANTAGES OF THE CURRENT ART

The main disadvantage with primer walking is the resynthesis of the walking primer after each round of sequencing. Studier (J Kieleczawa et al, Science 258, p1787, (1992)) and others (T Azhikina et al, PNAS, 90, p11460, (1993)) have proposed elegant schemes using modular primers constructed out of presynthesised sets to avoid de novo primer synthesis after each round of sequencing. None of these walking primer schemes, however, are suited to cycle sequencing.
Cycle sequencing requires primers that have annealing temperatures near to the optimal temperature for the polymerase enzyme used for the cycle sequencing. Primers between 18 and 24 residues long are generally used for cycle sequencing.
As the length of the primer increases, then the size of any presynthesised walking primer set needed in order to achieve a given probability of obtaining a perfect match within a given number of contiguous overlapping frames also increases. For primers between 18 and 24 residues long, the size of the presynthesised walking primer set required makes primer walking an impractical proposition.


ADVANTAGES OF THE CURRENT INVENTION

The current invention allows the use of a manageably sized set of presynthesised walking primers for cycled sequencing.


THE CURRENT INVENTION

The invention provides a method for primer walking sequencing of a nucleic acid target, which method comprises performing a series of sequencing reactions, each involving hybridising a primer to the target and effecting chain extension/chain termination of the primer, wherein for each sequencing reaction there is used a primer selected from a presynthesised set of walking primers of which the annealing temperatures are raised and/or the annealing properties improved without increasing their sequence complexity.
The invention also provides a library of y oligonucleotides, where y is defined as from 2 to 20000, each oligonucleotide comprising n nucleotide residues N and x nucleotide analogue residues X wherein base pairing with two or more of A C G and T, or forming stronger base interactions than A C G T, residues X in the oligonucleotide is defined as: either random or the residue X at the 5'-end of the oligonucleotide chain.
A DNA sequence is herein considered as a series of contiguous overlapping identically sized frames of residues with each frame translated from the next by one residue.
The following examples will define the terminology used herein:
Let each frame be n residues long:
Let there be i such frames suitable for selecting the walking primer
For each frame of n residues, the number of possible sequences is given by
The values of 4.sup.n for n between 5 and 9 are given below


______________________________________ n 4.sup.n ______________________________________ 5 1,024 6 4,096 7 16,384 8 65,536 9 262,144 ______________________________________
Hence the sequence of a given 5 mer will occur about once every 1 kb on average in random sequence DNA, the sequence of a given 6 mer will occur about once every 4 kb on average, the sequence of a given 7 mer will occur about once every 16 kb on average, the sequence of a given 8 mer will occur about once every 65 kb on average and the sequence of a given 9 mer will occur about once every 262 kb on average.
Thus one would expect a given 5 mer to form a perfect match at a unique location within a template of less than about 1 kb. For templates lar

REFERENCES:
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Jones et al., Nucleic Acids Res. 26(11), 2824-2826, 1998.
Hardin et al., Genome Res. 6(6), 545-550, 1996.
Azhikina et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 11460-11462 (1993).
Hou et al., Analytical Biochemistry 221, 136-141 (1994).
Kaczorowski et al., Analytical Biochemistry 221, 127-135 (1994).
Kotler et al., BioTechniques, vol. 17, No. 3 (1994).
Shen et al., BioTechniques, vol. 15, No. 1 (1993).

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