Method of detection of SP-A2 gene variants useful for prediction of predisposition to aspergillosis
a technology of aspergillosis and gene variants, applied in the field of detection of spa2 gene variants, can solve problems such as morbidity and mortality
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example 1
Identification of Allelic Variants of SP-A2 Gene
[0056] This example describes the identification of allelic variant of human surfactant protein A 2 gene by PCR and sequencing using certain oligonucleotide primers. According to the invention DNA was extracted from human peripheral blood leukocytes using a modification of salting out procedure. The concentration of the DNA was determined by measuring the optical density of the sample, at a wavelength of 260 nm. The DNA was then amplified by PCR by using the oligonucleotide primers:
(SEQ. ID. No. 1)5′ TGC CTG GAG CCC CTG GTG TCC CTG GAG AGC 3′(Forward)(SEQ. ID. No.2)5′ TGC CTC GTC CGC ATT CAC CCT TCA GAC TGC 3′(Reverse).
[0057] The sample were denatured at 95° C. for 5 minutes followed by 28 cycles of denaturation (95° C., 1 minutes), annealing (70° C., 1 minute), extension (72° C., 1 minute) and a final extension of 7 minutes at 72° C. in a PE GeneAmp PCR System 9600. This reaction produced a DNA fragment of 459 bp when analysed by ...
example 2
Nucleotide Sequence of the Allelic Variant of SP-A2 Gene
[0058] The nucleotide seq. of the allelic variant of SP-A 2 gene derived using the method as described in example 1.
[0059] In the above sequence the 2 SNP's as given in table II are at nucleotide position 1649 and 1660.
example 3
Patients with A Allele at 1660 Position are at Nearly Zero Risk for the ABPA Disease
[0060] A method as described in example 1 is applied to a series of DNA samples extracted from ABPA positive individuals and normal controls. There is observed a statistically significant difference (At position 1649 p=0.1193 and at position 1660 p=0.0156) in the frequency distributions of the SNP haplotypes generated using SNP in normal and ABPA patient SP-A2 chromosome. The results obtained are summarized in table below
TABLE IVSNP (G vs C) atSNP (G vs A) at1649 position1660 positionODDS RATIO (ABPA2.12644.7778patient vs Normal)Chi-square2.4265.846p-value0.11930.0156
[0061] A strong association of G (at 1649 position) and G (at 1660 position) haplotypes with ABPA disease chromosome indicated that SP-A2 alleles with the G (at 1649 position) and G (at 1660 position) haplotypes are predisposed to the disease. Therefore, these SNP haplotypes in the human SP-A2 gene could be used as a method of establ...
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