‘Cold SSCP’: a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses

Hongyo, Tadashi; Buzard, Gregory S.; Calvert, Richard J.; Weghorst, Christopher M.

doi:10.1093/nar/21.16.3637

Cited by 323 publications

(191 citation statements)

References 20 publications

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“…After an initial 3 min denaturation at 951C, there followed 40 cycles of 951C (20 s), 561C (30 s) and 721C (40 s), with a final extension of 10 minutes at 721C. Cold SSCP analysis was performed by denaturing 10 ml of the PCR product as described elsewhere, 45 and assayed on a 20 Â 20 Â 0.08 cm 3 nondenaturing polyacrylamide gel (20%, 49:1) at 4B61C, 300 V for 24 h. Sequences of a few subjects with different shifting bands were determined on an ABI-3100 sequencer (figure not shown) and a C-T point substitution at nucleotide-69 (referred to position on mRNA map NM_000816) was identified. The data were submitted to the dbSNP and identification number rs4480617 was assigned.…”

Section: Genotyping Methodsmentioning

confidence: 99%

Gender-specific contribution of the GABAA subunit genes on 5q33 in methamphetamine use disorder

et al. 2003

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Family and twins studies have suggested that genetic factors are involved in the development of substance use disorders. Several unrelated case/control association studies have reported associations of the GABA A subunit genes on 5q33 with the development of alcohol dependence. We hypothesized that these particular GABA A subunit genes also contribute to the development of methamphetamine use disorder. To test our hypothesis, we recruited cases using a series of questionnaires. Among the polymorphic SNPs, significant differences between cases and controls were identified in the female sample in the rs2279020 of the GABA A a1 subunit gene, and the novel SNP rs4480617 in the GABA A g2 subunit gene. No associations were found in the male sample. Further haplotype analysis identified several marker blocks significantly associated with methamphetamine use disorder in females; each block consists of the rs4480617. Our study provides preliminary evidence that the GABA A subunit genes on 5q33 may preferentially contribute to methamphetamine use disorder in females. There is increasing evidence that substance use disorders are familial and that genetic factors explain a substantial degree of the familial aggregation. 3 Grove et al 4 studied a sample of 32 pairs of monozygotic twins reared apart, and reported that inherited factors explained 45% of the variance in drug abuse and dependence. Pickens et al 5 found that the variance attributable to genetic factors in abuse of illicit drugs is 31% in males and 22% in females. Similarly, Tsuang et al 6 reported that genetic factors account for 34% of the variance in the individual's risk of developing a drug use disorder; for stimulant abuse, 44% of the variance is attributable to genetic factors. The pairwise concordance rate for stimulant abuse was 14.1% for monozygotic twins and 5.3% for dizygotic twins. 7 The supporting evidence is also derived from animal studies. For example, knockout of the dopamine D4 receptor gene in mice result in animals that are supersensitive to several substances, including methamphetamine. 8 For the GABA A a1 knockout mice, administration of amphetamine sulfate caused an

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Section: Genotyping Methodsmentioning

confidence: 99%

Gender-specific contribution of the GABAA subunit genes on 5q33 in methamphetamine use disorder

et al. 2003

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“…22 Primer sequences for PCR amplification of the 9 exons of the T␤R-I gene were described previously. 17 PCR was performed in a volume of 20 l containing 500 nM of forward and reverse primers and 2 units of HotStarTaq DNA polymerase (Qiagen, Valencia, CA).…”

Section: Pcr-sscp and Rflpmentioning

confidence: 99%

An intronic variant of the TGFBR1 gene is associated with carcinomas of the kidney and bladder

Chen

Jackson

Costello

et al. 2004

Intl Journal of Cancer

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TGF-␤ signaling is frequently perturbed in many human cancers, including renal cell carcinomas (RCCs) and transitional cell carcinomas (TCCs) of the bladder. Genetic alterations of the TGF-␤ type 1 receptor (TGFBR1) may contribute to these perturbations. We therefore examined variations in the TGFBR1 gene by PCR, SSCP and RFLP in carcinomas of the urinary system and in tissues from noncancer, age-matched controls. A G3 A variant 24 bp downstream of the exon/intron 7 boundary of the TGFBR1 gene (Int7G24A) was evident in patients with RCC (46.5%, n ‫؍‬ 86) and bladder and upper urinary tract TCC (49.2%, n ‫؍‬ 65) significantly more frequently than in age-matched controls (28.3%, n ‫؍‬ 113, p < 0.002 by 2 test). Moreover, 8 homozygous variant carriers were found in the cancer groups, whereas not a single homozygous variant carrier was found in the control group. The Int7G24A allele (both heterozygous G/A and homozygous A/A carriers) was associated with increased RCC incidence (OR ‫؍‬ 2.20, 95% CI 1.22-3.96) and TCC incidence (OR ‫؍‬ 2.45, 95% CI 1.89 -3.16). One somatic mutation of serine to phenylalanine at codon 57 of the TG-FBR1 gene was confirmed in an upper urinary tract TCC. In conclusion, the Int7G24A variant in the TGFBR1 gene is significantly more frequent in patients with RCC and TCC than normal age-matched controls, suggesting that it may represent a risk factor for the development of kidney and bladder carcinomas. © 2004 Wiley-Liss, Inc. Key words: TGF-␤ type 1 receptor; renal cell carcinoma; bladder transitional cell carcinoma; upper urinary tract transitional cell carcinoma; genetic variant; SSCPCancer of the urinary system is among the 5 most frequent malignancies in the United States based on recent cancer statistics. 1 More than 7% of all cancers in 2004 will be cancers of the kidney or urinary bladder. RCCs, which kill nearly 35% of afflicted patients, account for the majority of kidney neoplasms. TCC of the bladder and upper urinary tract is the fourth most frequent malignancy in men. 1 Advances in the genetic understanding of renal neoplasms have been made through successful cloning and mutational screening of the VHL, TSC and MET proto-oncogenes. However, many sporadic RCCs lack evidence of involvement of these 3 genes. Thus, other genes involved in cell growth regulation may be associated with sporadic RCC. Acquisition of TGF-␤ resistance has been suggested to be an important progression factor in human RCC and bladder TCC. 2,3 Disruption of the TGF-␤ signaling pathway has been hypothesized as the predominant mechanism through which many types of tumor cells gain a selective growth advantage. 4 We focused on the TGFBR1 gene, an important component of the TGF-␤ signaling pathway, for genetic analysis in cancers of the kidney and urinary bladder.The TGF-␤ signal is transduced by 2 transmembrane serinethreonine kinase receptors. TGF-␤ binds first to TGFBR2 homodimers, which then form heterotetramers with 2 TGFBR1 molecules. As a consequence, the TGFBR2 kinase phosphorylates and activates TGFBR1. ...

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“…The following oligonucleotides were synthesized as partially intron-based PCR primers of p53 and K-ras as described previously (Hongyo et al, 1995). Forty cycles of PCR ampli®cation were performed as follows: denaturation at 948C for 1 min, annealing at 608C for 1 min for exons, 5, 6 and 7 of p53; at 558C for exon 8 of p53, and at 508C for exon 1 of K-ras, and then extension at 728C for 1.5 min followed by a ®nal extension at 728C for 7 min.…”

Section: Pcr ± Sscp and Dna Sequencingmentioning

confidence: 99%

A novel molecular staging protocol for non-small cell lung cancer

et al. 1999

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A molecular staging protocol using reliable markers is of importance in predicting the prognosis of patients with non-small cell lung cancer (NSCLC) and for instituting their appropriate post-surgical treatment. We analysed tumor tissues from 187 NSCLC patients. The DNA and mRNA were extracted from frozen specimens, and then polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to investigate mutations of p53 from exons 5 ± 8, and mutations of K-ras at exon 1. To determine MRP-1/CD9 gene and KA11/CD82 gene expression, which have been postulated to be metastasis suppressor genes, we have applied quantitative RT ± PCR. A Cox multivariate regression analysis showed that nodal status, MRP-1/CD9 and K-ras status were signi®cant factors for prognosis (P50.0001, P=0.0083 and P=0.0004, respectively). Based on these results, we classi®ed the patients into three groups according to their MRP-1/ CD9 and K-ras status. Patients with both MRP-1/CD9 positive and wild K-ras tumors were de®ned as group A, patients with either reduced MRP-1/CD9 or mutant Kras tumors were de®ned as group B and patients with both reduced MRP-1/CD9 and mutant K-ras tumors were designated as group C. This new classi®cation was signi®cantly correlated with the tumor status and pathological stage (P=0.0098 and P=0.0017, respectively). The overall survival rate of the group A patients was signi®cantly better than the group B patients (59.6% vs 27.9%, P=0.0001) and also that of group B patients was better than the group C patients (27.9% vs 20.0%, P=0.0378). This tendency was also found in patients with 110 node-negative NSCLCs (A vs B vs C=75.8% vs 34.9% vs 0.0%, P50.0001). A Cox multivariate regression analysis in NSCLC patients demonstrated that an evaluation for both MRP-1/CD9 expression and K-ras mutations had a signi®cant prognostic e ect as well as nodal status (P50.0001).

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‘Cold SSCP’: a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses

Cited by 323 publications

References 20 publications

Gender-specific contribution of the GABAA subunit genes on 5q33 in methamphetamine use disorder

Gender-specific contribution of the GABAA subunit genes on 5q33 in methamphetamine use disorder

An intronic variant of the TGFBR1 gene is associated with carcinomas of the kidney and bladder

A novel molecular staging protocol for non-small cell lung cancer

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