Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1993-05-14
1996-06-11
Jones, W. Gary
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 536 243, 536 235, C12P 1934, C12Q 168, C07H 2104
Patent
active
055254926
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/GB91/01935, filed on Nov. 5, 1991, which is claiming priority to GB 9024005.2, filed on Nov. 5, 1990.
The present invention relates to a process for amplifying a desired nucleic acid sequence, and in particular for the amplification of a nucleic acid sequence encoding at least part of a human leucocyte antigen.
Human leucocyte antigen (HLA) allele typing has traditionally been performed by serological methods. This involves isolating white blood cells that express human leucocyte antigens on their surface and determining (usually microscopically) whether certain antibodies kill the cells.
Recently nucleic acid amplification techniques such as the polymerase chain reaction (PCR) has been used in combination with Southern blot techniques to type HLA class II alleles. Since there are a large number of cross-hybridising HLA class I sequences restriction fragment length polymorphism analysis has so far failed to distinguish HLA class I alleles and no report of successful HLA class I typing using PCR has appeared.
PCR techniques themselves are known from EP-A-0200362, EP-A-0201184 and EP-A-0258017, all of which are in the name of Cetus Corporation. These publications disclose processes for amplifying a desired nucleic acid sequence by treating separate complementary strands of nucleic acid with a molar excess of two oligonucleotide primers, and extending the primers (often using Klenow fragment) to form complementary primer extension products which act as templates for synthesising the desired nucleic acid sequence. EP-A-0258017 teaches that the use of DMSO in an amplification buffer is undesirable when using a heat stable polymerase such as Taq polymerase as it inhibits the activity of the polymerase, and that the addition of EDTA will halt amplification by inactivating the polymerase.
Although various nucleic amplification techniques are known in the art, successful amplification of a desired sequence will usually depend upon the primers employed to generate the extension products. However, even when the desired sequence is known, and a primer capable of hybridizing to that sequence determined, successful amplification is not guaranteed. Indeed, except within carefully chosen parameters, it is virtually impossible to predict whether a chosen primer will allow amplification. This unpredictability stems from the following two problems associated with amplification.
Firstly, the primer may loop back on itself (to form a hairpin) or form a complex structure, both of which will prevent amplification of the desired sequence.
Secondly, the primer may hybridize to a different sequence from the one that is desired to amplify. (This problem is also encountered when designing probes for a specific sequence.)
These factors contribute to the difficulties encountered with HLA typing.
HLA is the human version of the major histocompatibility complex (MHC) which is a cluster of genes that encode for cell surface antigens on leucocytes. These antigens are responsible for rejection of skin and organ grafts between individuals. Such antigens can be classified into three classes:
The present invention seeks to overcome or at least mitigate some or all of the above problems, and provide a PCR technique that may allow the typing and sub-typing of human leucocyte antigens.
Therefore, according to a first aspect of the present invention there is provided a process for amplifying a nucleic acid sequence comprising two complementary strands (which may or may not be hybridized), at least one strand coding for a human leucocyte antigen (HLA) sequence, the process comprising: acid sequence to be amplified, each primer being complementary or substantially complementary to a respective strand of the nucleic acid sequence; containing a polar aprotic solvent such as dimethylsulphoxide (DMSO) so that each extension product, when it is separated from its complement, can serve as a template for synthesis of an extension product of the other primer, at least one of the extension products comprising the HLA sequence
REFERENCES:
patent: 5130238 (1992-07-01), Malek et al.
Choo et al (Human Immunology 21:209-219) 1988.
Weiss et al (Immunobiology 170:367-380) 1985.
The Lancet vol. 337, No. 8742, Mar. 16, 1991, pp. 640-642, Hill et al.
The American Society for Histocompatibility & Immunogenetics, 16th Annual Meeting Abstracts, Nov. 3-7, 1990.
Bednarek Michael D.
Campbell Eggerton
ISIS Innovation, Ltd.
Jones W. Gary
LandOfFree
Process for amplifying HLA sequences does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for amplifying HLA sequences, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for amplifying HLA sequences will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-351073