Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-08-05
1999-06-08
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 231, 536 243, 525 5411, 428403, C12Q 168, C07H 2102, C08G 6348, B32B 516
Patent
active
059104066
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method and apparatus for carrying out manipulations on nucleic acid sequences using a solid support system, as well as to supports for use in the method and apparatus.
Various procedures are known for manipulating nucleic acid sequences. For example, EP-A-0 200 362 (Cetus) describes a liquid phase process for amplifying, detecting and/or cloning nucleic acid sequences. The process comprises the following steps
(i) treating separate complementary strands of the nucleic acid sequence with two oligonucleotide primers each of which hybridises to one of the strands;
(ii) extending the primers to form double stranded nucleic acid sequences;
(iii) denaturing the product from (ii) to produce single strands of nucleic acid; and
(iv) using the single strands from (iii) in a repeat of steps (i)-(iii) the overall procedure being repeated as often as necessary.
It is a feature of this prior technique that both complementary strands are used as templates for the second and further amplification steps. Furthermore in the method of EP-A-0 200 362 the reactant mixture includes unhybridised target nucleic acid, unhybridised copy target, and unhybridised primer which makes the system very inefficient. Additionally any mistake which occurs in the copying at any stage results in the mistake being copied into the "chain reaction".
Amplification techniques using solid phase support systems are also known, for example DYNAL (Trade Mark) magnetic beads as disclosed in EP-A-0 091 453 and EP-A-0 106 873. In use of such beads, DNA is synthesised onto a magnetic bead and then cleaved therefrom by ammonium hydroxide. The beads may then be separated from the synthesised DNA using a magnet. Alternatively, biotin may be added to one end of a synthesised or natural DNA sequence and the sequence recovered using a magnetic bead which has been pre-conjugated to streptavidin (thereby attracting the biotin to recover the DNA). A problem with this type of solid support is that the biotin streptavidin linkage is biodegradable.
Other known solid support systems include porous silicas. The presence of the pores can create problems since nucleotide chain growth occurs within the pores, resulting in inefficient washing and residues remaining within the pores, again reducing the yield and resulting in relatively inefficient coupling.
Ep-A-0 184 056 describes a method for the large scale production of DNA probes using a solid substrate. The process of this prior specification comprises covalently linking to a solid substrate a polynucleotide complementary to the probe to be produced and then hybridising the polynucleotide with an oligonucleotide which acts as a primer. The oligonucleotide is then extended in a direction away from the substrate (using the polynucleotide as a template) thereby to produce an extended sequence complementary to the bound polynucleotide. The hybridised polynucleotide and extended oligonucleotide are then denatured so as to release the extended oligonucleotide from the solid substrate for collection. The extended oligonucleotide may be used as an analytical probe. Thus, for example, the polynucleotide originally bound to the support may be a gene, and the extended oligonucleotide may be used as a probe for detecting the presence of that gene in a biological sample.
There are however a number of disadvantages associated with the method described in EP-A-0 184 056. In particular, i f the polynucleotide to be bound to the support is not "pure" and contains other polynucleotides then these will also become bound to the support. As a result, the oligonucleotide may also hybridise to these other polynucleotides so that the extended oligonucleotide ultimately obtained may in fact be a mixture of products. Therefore, the method is not particularly good for producing "pure" samples of nucleic acid.
It is therefore an object of the present invention to obviate or mitigate the abovementioned disadvantages.
According to a first aspect of the present invention there is provided a method of effecting a ma
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Hultman et al., Biotechniques 10(1) :84-93 (1991).
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Horvath et al. Methods in Enzymology 154: 314-326 (1987).
Jones W. Gary
Tepnel Medical Limited
Whisenant Ethan
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