Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support

Hultman, Thomas; Ståhl, Stefan; Homes, Erik; Uhlén, Mathias

doi:10.1093/nar/17.13.4937

Cited by 716 publications

(338 citation statements)

References 18 publications

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“…Capturing of the amplified DNA on a solid support comprises a simple and efficient single-step purification of the DNA from the PCR mixture. Solid phase techniques are applicable to automatic sequencing procedures, as shown recently by Hultman et al and Voss et al [19,20]. We conclude that nucleotide sequencing of affinity-captured PCR-amplified DNA fragments is a promising approach to the routine diagnosis of genetic disorders.…”

Section: Conclusion and Prospectssupporting

confidence: 59%

Direct sequencing of affinity‐captured amplified human DNA application to the detection of apolipoprotein E polymorphism

et al. 1989

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We describe a method for the direct sequencing of DNA amplified by the polymerase chain reaction (PCR). Biotin is introduced into one strand of the amplified DNA using a S-biotinylated PCR primer. The synthexised fragment is captured on an avidin-matrix and rendered single stranded, whereafter the nucleotide sequence of the immobilized strand is determined by the chain termination method. The method involves few and simple operations and is thus applicable to the analysis of human genes for routine diagnostic purposes. Here we applied the method for determination of the three-allelic polymorphism of the apolipoprotein E (apo E) gene. We were able to correctly identify the alleles in both homoxygous and heteroxygous samples.

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Section: Conclusion and Prospectssupporting

confidence: 59%

Direct sequencing of affinity‐captured amplified human DNA application to the detection of apolipoprotein E polymorphism

et al. 1989

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“…1, indicated that the patient had no apparent deletion in her Y chromosome ( Table 2). The PCR coupled with the direct solid phase sequencing of the conserved region of SRY with magnetic beads as solid support [7] revealed that her SRY gene had a 100% nude- otide sequence identity with the reported cloned sequence [1].…”

Section: Case Reportmentioning

confidence: 96%

Intact Sex Determining Region Y (SRY) in a Patient with XY Pure Gonadal Dysgenesis and a Twin Brother.

et al. 1994

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Abstract.A patient with an apparently normal 46, XY karyotype, suffering from pure gonadal dysgenesis and of short stature was investigated. The patient, who was growth retarded, was a 30-yearold married Japanese woman with a history of primary amenorrhea and infertility with a weight of 42 kg and a height of 146 cm. She has a phenotypically and karyotypically normal dizygotic twin brother with normal development. Southern-blot and polymerase chain-reaction analyses revealed no apparent deletions in the patient's Y chromosome, including the sex-determining region Y (SRY). The DNA sequencing of the SRY gene showed a 100% nucleotide sequence identity with the reported cloned sequence. Sex reversal in the present case may be due to mutation at a locus other than SRY in the sex determining pathway, a gene potentially involved in the determination of human constitution.

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“…The biotinylated amplicons were immobilized onto streptavidin-coated solid support and strand speci®c elution with 0.05 M NaOH produced a clean template for sequencing. Solid-phase sequencing was performed as published (Berg et al, 1995;Hultman et al, 1989) with automated laser¯uorescent analysis (ALF TM , Pharmacia Biotech AB, Uppsala, Sweden). The sequences were compared with wild type p53 sequence as exempli®ed in Figure 4.…”

Section: Solid-phase Sequencingmentioning

confidence: 99%

Molecular pathology in basal cell cancer with p53 as a genetic marker

et al. 1997

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Human basal cell cancer (BCC) has unique growth characteristics with virtual inability to metastasize. We investigated clonality and genetic progression using p53 mutations as marker. Sampling was done through microdissection of frozen immunohistochemically stained 16 mm slices of tumors. From 11 BCC tumors 78 samples were analysed. Direct DNA sequencing of exons 5 ± 8 was performed, haplotypes were determined after cloning of p53 exons and loss of heterozygosity (LOH) ascertained by microsatellite analysis. All tumors had p53 mutations and in a majority both p53 alleles were a ected, commonly through missense mutations. Microdissection of small parts (50 ± 100 cells) of individual tumors showed BCC to be composed of a dominant cell clone and prone to genetic progression with appearance of subclones with a second and even third p53 mutation. Samples from normal immunohistochemically negative epidermis always showed wild type sequence, except for a case of previously unknown germline p53 mutation. Our analysis also included p53 immunoreactive patches i.e. morphologically normal epidermis with a compact pattern of p53 immunoreactivity. Mutations within those were never the same as in the adjacent BCC. This detailed study of only one gene thus uncovered a remarkable heterogeneity within a tumor category famous for its benign clinical behavior.

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Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support

Cited by 716 publications

References 18 publications

Direct sequencing of affinity‐captured amplified human DNA application to the detection of apolipoprotein E polymorphism

Direct sequencing of affinity‐captured amplified human DNA application to the detection of apolipoprotein E polymorphism

Intact Sex Determining Region Y (SRY) in a Patient with XY Pure Gonadal Dysgenesis and a Twin Brother.

Molecular pathology in basal cell cancer with p53 as a genetic marker

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