Co-dominant genetic diagnosis test

Details Co-dominant genetic diagnosis test Co-dominant genetic diagnosis test

1998-08-04

2001-05-15

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S024310, C536S024330, C536S063000, C424S085100

Reexamination Certificate

active

06232063

ABSTRACT:

The present invention relates to a co-dominant genetic diagnosis test, i.e., a test to distinguish homozygous and heterozygous individuals for a polymorphous allele in a population.
A rapid and effective method for detecting a point mutation at the genomic DNA level is essential to the identification of polymorphisms both for genetic studies and predicting a risk of disease linked to that polymorphism, and for the study of the molecular bases for hereditary diseases. It is also essential for the development of a genetic diagnosis test.
In the remainder of the text, the term “point mutation” is used to encompass a change in a sequence, be it a nucleotide transition, transversion, deletion or addition; more generally, this includes transitions, transversions, deletions or additions of 1 to 6 nucleotides.
More generally, such a type of rapid, effective and inexpensive detection method can be applied to detecting mutations in any living organism, be it a micro-organism, animal or plant, the method being of particular importance for diploid organsims. Such a type of detection is applicable to the fields of agriculture and food, medicine or veterinary diagnoses, or to animal or plant selection.
PCR (1) represented a great advance for genomic DNA analysis. That technique enables genetic diseases to be diagnosed when combined with other techniques (2, 3, 4, 5, 6, 7, 8, 9); it may be a combination of PCR and direct sequencing (2, 3, 4, 10) or the allele specific oligonucleotide (ASO) technique (11, 12).
In some cases, the appearance of a point mutation can create or destroy a recognition site in a restriction enzyme (13); the presence or absence of the restriction site can be used to cany out diagnoses, as has recently been demonstrated for sickle-cell anaemia diagnosis (7); in the same way, a restriction polymorphism can be linked to a non characterised mutation which enables a diagnosis to be made in families by analysis of that polymorphism after amplification. A number of techniques have been developed with the aim of enabling such mutations to be detected by combining PCR with other types of reaction. These are in particular:
the technique known as PCR amplification of specific alleles (PASA), a modification of the PCR technique using either an oligonucleotide primer which hybridises with the wild allele but does not hybridise with the mutant allele, or vice versa: the amplified product will thus be specific for the allele for which the primers have been selected and amplification is thus ineffective if the primer has not hybridised with the corresponding allele (14);
HEIM et al. (15) used a set of different primers to amplify the two alleles, the amplifications being followed by allele specific PCR;
SCHUSTER et al. (16) combined asymmetric PCR with allele specific PCR using a set of 3 oligonucleotide primers in a single reaction mixture to detect a point mutation in the apoB gene; however, for recessive diseases, that (simple) technique cannot distinguish individuals carrying a single mutated allele from diseased individuals with two mutated alleles.
Other methods have been used, particularly for demonstrating the creation of a restriction site by mutation from an amplification product. That technique has been used to detect haemophilia B (17) or haemophilia A (18).
None of the diagnosis test techniques described is suitable for widespread use in a population as they require either lengthy, complex, and expensive steps which necessitate the exercise of a high level of technical skill, or they cannot differentiate homozygous from heterozygous individuals; other techniques such as allele specific oligonucleotides are dominant types and cannot differentiate a homozygous individual from a heterozygous individual having one normal allele and one mutated allele. For genetic diagnosis, in particular predicting the risk of genetic disease in given populations, it is extremely important to be able to identify those two populations without resorting to complex and expensive techniques.
The present invention can overcome the disadvantages of the different techniques described in the literature, and in particular it avoids the use of radio-elements; it concerns a method of detecting the homoygous or heteroygous state of a mutation assumed to be present in a nucleic acid, characterised in that two nucleic acid amplifications are used, the two amplifications respectively requiring the use of at least two primer pairs:
the first pair being constituted by an oligonucleotide which is specific for the wild allele (A) and a second oligonucleotide (B) which is different from (A);
the second pair being constituted by an oligonucleotide which is specific for the mutant allele (A′) and a second oligonucleotide (C), which is different from (A′) and from (B);
the difference in length between the amplified fragments between (A) and (B) and between (A′) and (C) being sufficient to be detected by conventional analysis techniques.
These two amplifications are simultaneous.
The term “simultaneous” here means that the reaction products are simultaneously analysed using conventional methods, in particular analytical or preparative DNA separation, especially polyacrylamide gel or agarose gel electrophoresis; however, it will be clear to the skilled person that any other method of analysing the size of the amplified fragments (such as chromatography) can be considered as an equivalent means in the method of the invention.
The two amplification reactions can be carried out either in two different reaction mixtures if (A) and (A′) are complementary to the same DNA chain, or they can be carried out in the same reaction mixture if (A) and (A′) are complementary to the (+) and (−) strands of the DNA respectively.
In particular, the differences in the lengths can be detected by the existence of two different bands after agarose gel electrophoresis migration, for example; however, it will be clear that when detection methods become more sensitive, the differences in the lengths between the amplified fragments can be reduced.
More generally, any technique for amplifying a DNA sequence which comprises the use of at least two primers and a polymerase to synthesise the complementary sequence between the two primers, whatever the improvements thereto, can be used to implement the invention which resides in the simultaneous use of two different primer pairs and simultaneous visualisation of the PCR products.
The two primer pairs, (A) and (B) and (A′) and (C), can be symmetrical or inverted, in other words (A) and (A′) are hybridisable with the same strand of the double helix, and (B) and (C) with the other strand, or in contrast, the (A) (B) pair and the (A′) (C) pair can be inverted, i.e., (A) and (A′) are hybridisable with complementary strands of the DNA chain, like (B) and (C).
In the first variation, the two amplifications carried out by the two primer pairs must be carried out separately, then the reaction products must be mixed for analysis using conventional methods.
In contrast, in the second variation, the reaction products can be mixed from the start, since amplification between (A) and (C) cannot occur. However, in the latter case, primers (A) and (A′) must have a sufficiently different sequence to avoid unwanted hybridisation between (A) and (A′) in the reaction mixture. The only requirement is that (A) and (A′) carry the nucleotide corresponding to that for which the mutation is sought.
Finally, this technique can be used whatever the DNA-containing organism, be it a micro-organism, bacterium, virus, animal or plant; however it is of particular importance for diploid or polyploid organisms.
The usefulness of this novel technique has been demonstrated by identifying a mutation in Amish populations from southern Indiana carrying a gene coding for a protein involved in a recessive autosomal disease: limb-girdle muscular dystrophy.
While the conventional PCR method has proved to be extremely powerful for amplifying target sequences,

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