Detection of heterozygous mutations in the RB1 gene in retinoblastoma patients using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing

A. Hogg, Z. Onadim, P. N. Baird, John Kenneth Cowell

Research output: Contribution to journalArticle

77 Citations (Scopus)

Abstract

Several families segregating the autosomal dominant form of the hereditary retinoblastoma predisposition gene have been analysed for the causative mutation. We have used the single-strand conformation polymorphism (SSCP) technique to screen for mutations, exon by exon, in the RB1 gene in affected patients from these families. The SSCP technique has proved a rapid and simple technique which relies on the sequence-dependent migration of single-stranded DNA in a non-denaturing polyacrylamide gel. Oligonucleotide primers flanking all 27 exons and the promoter region of the RB1 gene are reported here. The polymerase chain reaction (PCR)-amplified products range in size from 212 to 625bp and include a flanking intron sequence which allows detection of mutations in these regions. The sensitivity of SSCP is optimal when DNA fragments are approximately 200bp long. Consequently, restriction enzyme sites for each amplified region were identified, reducing the size of the PCR products analysed to less than 250bp. Bands with aberrant migration patterns were observed on SSCP gels in the lymphocyte DNA from two patients with bilateral, familial retinoblastoma. Sequence analysis of these DNA fragments revealed the causative mutations. These consisted of a 1-bp insertion of a T in the coding strand of exon 20 and a G→A mutation in the coding strand of exon 14. This approach has proved to be a powerful method for the rapid detection of germline mutations in the RB1 gene, a programme which can be extended to individuals with new mutations.

Original languageEnglish (US)
Pages (from-to)1445-1451
Number of pages7
JournalOncogene
Volume7
Issue number7
StatePublished - Jan 1 1992
Externally publishedYes

Fingerprint

Retinoblastoma Genes
Exons
Polymerase Chain Reaction
Mutation
Retinoblastoma
Genes
DNA Primers
Germ-Line Mutation
Single-Stranded DNA
DNA
DNA Sequence Analysis
Genetic Promoter Regions
Introns
Gels
Lymphocytes
Enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cancer Research

Cite this

Detection of heterozygous mutations in the RB1 gene in retinoblastoma patients using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing. / Hogg, A.; Onadim, Z.; Baird, P. N.; Cowell, John Kenneth.

In: Oncogene, Vol. 7, No. 7, 01.01.1992, p. 1445-1451.

Research output: Contribution to journalArticle

@article{e90d0e0ea93c4895b9d98a28c257dcee,
title = "Detection of heterozygous mutations in the RB1 gene in retinoblastoma patients using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing",
abstract = "Several families segregating the autosomal dominant form of the hereditary retinoblastoma predisposition gene have been analysed for the causative mutation. We have used the single-strand conformation polymorphism (SSCP) technique to screen for mutations, exon by exon, in the RB1 gene in affected patients from these families. The SSCP technique has proved a rapid and simple technique which relies on the sequence-dependent migration of single-stranded DNA in a non-denaturing polyacrylamide gel. Oligonucleotide primers flanking all 27 exons and the promoter region of the RB1 gene are reported here. The polymerase chain reaction (PCR)-amplified products range in size from 212 to 625bp and include a flanking intron sequence which allows detection of mutations in these regions. The sensitivity of SSCP is optimal when DNA fragments are approximately 200bp long. Consequently, restriction enzyme sites for each amplified region were identified, reducing the size of the PCR products analysed to less than 250bp. Bands with aberrant migration patterns were observed on SSCP gels in the lymphocyte DNA from two patients with bilateral, familial retinoblastoma. Sequence analysis of these DNA fragments revealed the causative mutations. These consisted of a 1-bp insertion of a T in the coding strand of exon 20 and a G→A mutation in the coding strand of exon 14. This approach has proved to be a powerful method for the rapid detection of germline mutations in the RB1 gene, a programme which can be extended to individuals with new mutations.",
author = "A. Hogg and Z. Onadim and Baird, {P. N.} and Cowell, {John Kenneth}",
year = "1992",
month = "1",
day = "1",
language = "English (US)",
volume = "7",
pages = "1445--1451",
journal = "Oncogene",
issn = "0950-9232",
publisher = "Nature Publishing Group",
number = "7",

}

TY - JOUR

T1 - Detection of heterozygous mutations in the RB1 gene in retinoblastoma patients using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing

AU - Hogg, A.

AU - Onadim, Z.

AU - Baird, P. N.

AU - Cowell, John Kenneth

PY - 1992/1/1

Y1 - 1992/1/1

N2 - Several families segregating the autosomal dominant form of the hereditary retinoblastoma predisposition gene have been analysed for the causative mutation. We have used the single-strand conformation polymorphism (SSCP) technique to screen for mutations, exon by exon, in the RB1 gene in affected patients from these families. The SSCP technique has proved a rapid and simple technique which relies on the sequence-dependent migration of single-stranded DNA in a non-denaturing polyacrylamide gel. Oligonucleotide primers flanking all 27 exons and the promoter region of the RB1 gene are reported here. The polymerase chain reaction (PCR)-amplified products range in size from 212 to 625bp and include a flanking intron sequence which allows detection of mutations in these regions. The sensitivity of SSCP is optimal when DNA fragments are approximately 200bp long. Consequently, restriction enzyme sites for each amplified region were identified, reducing the size of the PCR products analysed to less than 250bp. Bands with aberrant migration patterns were observed on SSCP gels in the lymphocyte DNA from two patients with bilateral, familial retinoblastoma. Sequence analysis of these DNA fragments revealed the causative mutations. These consisted of a 1-bp insertion of a T in the coding strand of exon 20 and a G→A mutation in the coding strand of exon 14. This approach has proved to be a powerful method for the rapid detection of germline mutations in the RB1 gene, a programme which can be extended to individuals with new mutations.

AB - Several families segregating the autosomal dominant form of the hereditary retinoblastoma predisposition gene have been analysed for the causative mutation. We have used the single-strand conformation polymorphism (SSCP) technique to screen for mutations, exon by exon, in the RB1 gene in affected patients from these families. The SSCP technique has proved a rapid and simple technique which relies on the sequence-dependent migration of single-stranded DNA in a non-denaturing polyacrylamide gel. Oligonucleotide primers flanking all 27 exons and the promoter region of the RB1 gene are reported here. The polymerase chain reaction (PCR)-amplified products range in size from 212 to 625bp and include a flanking intron sequence which allows detection of mutations in these regions. The sensitivity of SSCP is optimal when DNA fragments are approximately 200bp long. Consequently, restriction enzyme sites for each amplified region were identified, reducing the size of the PCR products analysed to less than 250bp. Bands with aberrant migration patterns were observed on SSCP gels in the lymphocyte DNA from two patients with bilateral, familial retinoblastoma. Sequence analysis of these DNA fragments revealed the causative mutations. These consisted of a 1-bp insertion of a T in the coding strand of exon 20 and a G→A mutation in the coding strand of exon 14. This approach has proved to be a powerful method for the rapid detection of germline mutations in the RB1 gene, a programme which can be extended to individuals with new mutations.

UR - http://www.scopus.com/inward/record.url?scp=0026718136&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026718136&partnerID=8YFLogxK

M3 - Article

C2 - 1352398

AN - SCOPUS:0026718136

VL - 7

SP - 1445

EP - 1451

JO - Oncogene

JF - Oncogene

SN - 0950-9232

IS - 7

ER -