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Development of low-density oligonucleotide microarrays for detecting mutations causing Wilson's disease.

Mathur M, Singh E, Poduval TB, Rao AV - Indian J. Med. Res. (2015)

Bottom Line: The hybridization reaction were found to be consistent across the surface of a given microarray.Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C.The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, India.

ABSTRACT

Background & objectives: Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in ATP7B, a copper transporter gene, leading to hepatic and neuropsychiatric manifestations due to copper accumulation. If diagnosed early, WD patients can be managed by medicines reducing morbidity and mortality. Diagnosis of this disease requires a combination of tests and at times is inconclusive due to overlap of the symptoms with other disorders. Genetic testing is the preferred alternative in such cases particularly for individuals with a family history. Use of DNA microarray for detecting mutations in ATP7B gene is gaining popularity because of the advantages it offers in terms of throughput and sensitivity. This study attempts to establish the quality analysis procedures for microarray based diagnosis of Wilson's disease.

Methods: A home-made microarrayer was used to print oligonucleotide based low-density microarrays for addressing 62 mutations causing Wilson's disease reported from Indian population. Inter- and intra- array comparisons were used to study quality of the arrays. The arrays were validated by using mutant samples generated by site directed mutagenesis.

Results: The hybridization reaction were found to be consistent across the surface of a given microarray. Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C. Our arrays have shown > 80 per cent sensitivity in detecting these 62 mutations.

Interpretation & conclusions: The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously. This study also provides information on some of the important parameters required for microarray based diagnosis of genetic disorders.

No MeSH data available.


Related in: MedlinePlus

(a). Magnified view of a scanned microarray (13.5 x 13.5 mm) hybridized to wild-type human genomic DNA sample [inset is shown in Fig. 2(b)]. 2(b). A portion of the array wherein, the spots corresponding to wild-type and mutant and probes are shown as underlined with solid or dashed lines, respectively. Perfect match (PM) probes are underlined by a continuous line while the adjoining mismatch (MM) probes are underlined by dotted lines. The forward and reverse complement probes and their corresponding mutant probes are indicated by arrows. Spot triples shown in boxed region correspond to exon-specific positive control probes.
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Figure 2: (a). Magnified view of a scanned microarray (13.5 x 13.5 mm) hybridized to wild-type human genomic DNA sample [inset is shown in Fig. 2(b)]. 2(b). A portion of the array wherein, the spots corresponding to wild-type and mutant and probes are shown as underlined with solid or dashed lines, respectively. Perfect match (PM) probes are underlined by a continuous line while the adjoining mismatch (MM) probes are underlined by dotted lines. The forward and reverse complement probes and their corresponding mutant probes are indicated by arrows. Spot triples shown in boxed region correspond to exon-specific positive control probes.

Mentions: Microarray hybridizations: Image of one of the microarrays hybridized to wild-type sample is shown in Fig. 2. It should be noted that the picture depicted here is a magnified portion of a 13.5 mm x 13.5 mm region on the slide. The first and last triple spots (Fig. 2A) are the positive control spots (DIG labelled oligo). The spot intensities varied from probe to probe even for the wild-type probes. However, for most of the probe-pairs, the perfect-match (PM) probes yielded higher intensities compared to the corresponding mismatch (MM) probes (Fig. 2B).


Development of low-density oligonucleotide microarrays for detecting mutations causing Wilson's disease.

Mathur M, Singh E, Poduval TB, Rao AV - Indian J. Med. Res. (2015)

(a). Magnified view of a scanned microarray (13.5 x 13.5 mm) hybridized to wild-type human genomic DNA sample [inset is shown in Fig. 2(b)]. 2(b). A portion of the array wherein, the spots corresponding to wild-type and mutant and probes are shown as underlined with solid or dashed lines, respectively. Perfect match (PM) probes are underlined by a continuous line while the adjoining mismatch (MM) probes are underlined by dotted lines. The forward and reverse complement probes and their corresponding mutant probes are indicated by arrows. Spot triples shown in boxed region correspond to exon-specific positive control probes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418154&req=5

Figure 2: (a). Magnified view of a scanned microarray (13.5 x 13.5 mm) hybridized to wild-type human genomic DNA sample [inset is shown in Fig. 2(b)]. 2(b). A portion of the array wherein, the spots corresponding to wild-type and mutant and probes are shown as underlined with solid or dashed lines, respectively. Perfect match (PM) probes are underlined by a continuous line while the adjoining mismatch (MM) probes are underlined by dotted lines. The forward and reverse complement probes and their corresponding mutant probes are indicated by arrows. Spot triples shown in boxed region correspond to exon-specific positive control probes.
Mentions: Microarray hybridizations: Image of one of the microarrays hybridized to wild-type sample is shown in Fig. 2. It should be noted that the picture depicted here is a magnified portion of a 13.5 mm x 13.5 mm region on the slide. The first and last triple spots (Fig. 2A) are the positive control spots (DIG labelled oligo). The spot intensities varied from probe to probe even for the wild-type probes. However, for most of the probe-pairs, the perfect-match (PM) probes yielded higher intensities compared to the corresponding mismatch (MM) probes (Fig. 2B).

Bottom Line: The hybridization reaction were found to be consistent across the surface of a given microarray.Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C.The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, India.

ABSTRACT

Background & objectives: Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in ATP7B, a copper transporter gene, leading to hepatic and neuropsychiatric manifestations due to copper accumulation. If diagnosed early, WD patients can be managed by medicines reducing morbidity and mortality. Diagnosis of this disease requires a combination of tests and at times is inconclusive due to overlap of the symptoms with other disorders. Genetic testing is the preferred alternative in such cases particularly for individuals with a family history. Use of DNA microarray for detecting mutations in ATP7B gene is gaining popularity because of the advantages it offers in terms of throughput and sensitivity. This study attempts to establish the quality analysis procedures for microarray based diagnosis of Wilson's disease.

Methods: A home-made microarrayer was used to print oligonucleotide based low-density microarrays for addressing 62 mutations causing Wilson's disease reported from Indian population. Inter- and intra- array comparisons were used to study quality of the arrays. The arrays were validated by using mutant samples generated by site directed mutagenesis.

Results: The hybridization reaction were found to be consistent across the surface of a given microarray. Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C. Our arrays have shown > 80 per cent sensitivity in detecting these 62 mutations.

Interpretation & conclusions: The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously. This study also provides information on some of the important parameters required for microarray based diagnosis of genetic disorders.

No MeSH data available.


Related in: MedlinePlus