Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-31
2002-12-31
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C536S023100, C536S023500, C536S024300, C536S024330
Reexamination Certificate
active
06500614
ABSTRACT:
The invention relates to a method and a kit for identifying an unknown allele of a polyallelic gene.
1. BACKGROUND TO THE INVENTION
1.1 General Introduction
Many genes exist an multiple alleles which differ from each other by small differences in sequence. It is sometimes desirable to identify an unknown allele of a polyallelic gene. For example, such identification is often necessary to match the alleles of the human leucocyte antigen (HLA) genes in a prospective donor and a prospective recipient in a tissue or organ transplant operation; if the donor and recipient have the same HLA alleles, the probability of the recipient rejecting the donor's tissue is greatly reduced.
However, it can be a difficult task to identify precisely an unknown allele of a polyallelic gene because two alleles can differ from each other by as little as one nucleotide.
The difficulties are increased in genes which have a very large number of different alleles, such as the major histocompatibility complex (MHC) genes (e.g. the HLA class I genes which have 222 known alleles).
Up to date the most favourable bone marrow transplant (BMT) and kidney transplant results have been obtained using sibling donors who are genotypically HU-identical to the recipient but such donors are available for only about 30% of patients
(1-5)
. BMT using unrelated donor s can be successful, but theme transplants have higher rates of graft failure, increased incidence and severity of Graft versus Host Disease and more frequent complications related to delayed or inadequate immune reconstitution
(4)
.
New molecular biological methods for detection of genetic polymorphism currently provide an opportunity to improve matching of unrelated donors as well as a research tool to investigate the relationship between genetic disparity and transplant complications. These molecular typing methods include sequence-specific amplification, hybridisation with oligonucleotide probes, heteroduplex analysis, single strand conformation polymorphism analysis and direct nucleotide sequencing.
Each of these molecular approaches has been used for routine HLA class II typing
(6)
, but a variety of reasons related to the HLA class I gene structure has complicated and made relatively unsuccessful their application to class I typing. The reasons for these complications are the extensive polymorphism of class I and the degree of sequence homology between the A, B and C loci of class I. In addition, sequence homology between class I classical and non-classical genes and the reported 12 pseudo genes can cause problems for specific locus amplifications
(7)
.
The low occurrence of “allele specific” sequences at polymorphic sites is a feature of the HLA class I genes. that has limited the resolution of all current DNA typing approaches. An “allele specific” sequence is a sequence that is only present in one allele and can therefore be used to distinguish the allele from other alleles. The occurrence on more than one exon of the specific sites for determining the allelic specificity causes additional problems in the identification of individual alleles. As a result, there is at present no single method of typing which can identify all HLA class I alleles of high resolution; see Table A below.
TABLE A
Comparison of some of the currently used DNA typing methods for HLA class I alleles
Method
Reference
HLA focus
Reagents
Resolution
SSO
Date et al ′96
A
91 probes
High
Tissue Antigens 47:93-101
SSO
Fernandez-Villa ′95
B
99
Medium-high
Tissue Antigens 45:153-168
SSO
Levine/Yang ′94
Cw
64 probes
Medium-high
Tissue Antigens 44:174-183
SSP
Bunce et al ′95
A, B, Cw
104 primer
Low (≧serology)
Tissue Antigens 46:355-367
mixes
SSP
Bunce/Welsh ′94
Cw
22 primer
Low (=serology)
Tissue Antigens 43:7-17
mixes
SSP
Krausa et al ′93
A9, 10, 19, 28
30 primer
Medium
Tissue Antigens 42:91-99
mixes
SSP
Krausa et al ′95
A2
15 primer
High
(nested)
Tissue Antigens 45:223-231
mixes
PCR-
Tatari et al ′95
Cw (partial, 23)
3 primer pairs,
High
RFLP
ProcNatlAcadSci 92:8803-7
11 endonucleases
SSP-
Blasczyk et al ′95
A (40 alleles)
27 primer pairs
High
SEQUENCING
Tissue Antigens 46:86-95
automatic sequencing
with fluorescent tracers
SSP-
Petersdorf/Hansen ′95
B (98 alleles)
5 primer pairs
Medium-high
SEQUENCING
Tissue Antigens 46:73-85
automatic sequencing
with fluourescent tracers
URSTO
Arguello et al ′96
A, B, Cw (201
3 primer pairs
High
ProcNatlAcadSci 9 3:10961-5
alleles)
40 probes
Methods For Allele Separation
1.2 Sequence Specific Primer Amplification (PCR-SSP)
This method utilises both the group-specific and, when present, allele-specific sequence sites in PCR primer design. The SSP design is based on the amplification refractory mutation system (ARMS), in which a mismatch at the 3′ residue of the primer inhibits non-specific amplification
(8,9)
.
Although each SSP reaction may not individually provide sufficient specificity to define an allele, the use of combinations of sequence specific primers allows the amplification of their common sequences to give the desired specificity.
However, despite its high accuracy, PCR-SSP is only in some cases more informative than serology. The reason for this is the low occurrence of allele specific sequence motifs in the exons and this limitation has stimulated a vast amount of research into the identification of allele specific motifs even in the intron sequences
(10)
. However, up to date this approach has not contributed considerably to the identification of more alleles.
Another limitation of this method is that it detects a limited number of polymorphic sequences which are utilised to predict the entire sequence. If an unknown allele is present in a particular sample this extrapolation may be incorrect.
In addition, the successful use of the technique relies on group specific amplification and therefore prior knowledge of broad HLA specificity is needed.
1.3 Single Strand Conformation Polymorphism (SSCP)
This technique is based on the electrophoretic mobility of single stranded nucleic acids in a non-denaturing polyacrylamide gel, which depends mainly on sequence-related conformation
(11-13)
. The technique can be employed for isolating single alleles which could then be used for further manipulation and analysis such as direct sequencing. The pattern of bands obtained after electrophoresis may be diagnostic for an allele
(14,15)
.
The major disadvantage of SSCP is the tendency of DNA single strand to adopt many conformational forms under the same electrophoretic conditions resulting in the presence of several bands from the same product; this makes the identification more difficult. In addition there is a high degree of variation and inconsistency in the sensitivity of this method for detecting mutations or allelic variations and there is a physical limitation in the size of the DNA fragment which is of the order of 200-400 base pairs (16).
1.4 Denaturing Gradient Gel Electrophoresis (DGGE) and Temperature Gradient Gel Electrophoresis (TGGE) (17.18)
The underlying principle of both techniques is the difference in the degree of melting between two alleles (double stranded DNA) which results in a reduction of mobility of the DNA fragments in polyacrylamide gels containing a denaturing reagent (DGGE) or a temperature gradient (TGGE).
Both techniques have been used frequently for screening mutations in genetic systems with one or two variants. They are only rarely used for the separation of alleles in highly polymorphic systems such as HLA.
Both techniques require specific conditions for a particular system under investigation and, in addition, where two alleles share common sequence segments with low melting points they may not always be differentiated. The simultaneous melting of both alleles will produce very similar retardations.
1.5. Cloning of DNA
This is the classical method of preparation of a single sequence, i.e. the sequence derived from a single allele. A variety of constructs has been used to introduce the required DNA fragment into a plasm
Arguello Rafael
Avakian Hovanes
Madrigal Alejandro
Horlick Kenneth R.
Medlen & Carroll LLP
Strzelecka Teresa
The Anthony Nolan Bone Marrow Trust
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