‘Touchdown’ PCR to circumvent spurious priming during gene amplification

Don, R. H.; Cox, Peter; Wainwright, Brandon; Baker, Kevin; Mattick, John S.

doi:10.1093/nar/19.14.4008

Cited by 2,402 publications

(1,350 citation statements)

References 4 publications

Supporting

Mentioning

1,313

Contrasting

Unclassified

Order By: Relevance

“…The ®rst PCR product was diltued 1 : 10 in double distilled sterile water and used for the second PCR reaction, which was performed in a 10 ml reaction volume containing 20 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 100 mM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), 0.5 mM of forward and reverse primer, 0.25 unit of AmpliTaq polymerase (GIBCO-BRL, Alameda, CA, USA), 0.25 ml of [a-32 P]dCTP (3000 Ci/mol, 10 mCi/ml Amersham LIFE SCIENCE, Arlington Heights, IL, USA), and 1 ± 2 ml of diluted ®rst PCR product. For both PCR reactions, a`touch-down' PCR (Don et al, 1991) was performed. After initial denaturation of 948C for 4 ± 8 min, 11 cycles each consisting of denaturation at 958C for 20 s, annealing at 65 to 568C for 55 s and extension at 728C for 20 s were performed, followed by additional 24 cycles which included denaturation at 908C for 20 s, annealing at 558C for 20 s and extension at 728C for 20 s. Then, a 5 ml aliquot of each second PCR reaction was diluted 1 : 4 with loading bu er, heat denatured, and separated by electrophoresis on a denaturing 6% polyacrylamide gel containing urea as published (Hung et al, 1995).…”

Section: Polymorphic Dna Markers and Pcr-loh Analysismentioning

confidence: 99%

Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma

et al. 1999

View full text Add to dashboard Cite

To understand the molecular pathways involved in the pathogenesis of squamous cell lung carcinoma, we obtained DNA from 94 microdissected foci from 12 archival surgically resected tumors including histologically normal epithelium (n=13), preneoplastic lesions (n=54), carcinoma is situ (CIS) (n=15) and invasive tumors (n=12). We determined loss of heterozygosity (LOH) at 10 chromosomal regions (3p12, 3p14.2, 3p14.1-21.3, 3p21, 3p22-24, 3p25, 5q22, 9p21, 13q14 RB, and 17p13 TP53) frequently deleted in lung cancer, using 31 polymorphic microsatellite markers, including 24 that spanned the entire 3p arm. Our major ®ndings are as follows: (1) Thirty one percent of histologically normal epithelium and 42% of mildly abnormal (hyperplasia/metaplasia) specimens had clones of cells with allelic loss at one or more regions; (2) There was a progressive increase of the overall LOH frequency within clones with increasing severity of histopathological changes; (3) The earliest and most frequent regions of allelic loss occurred at 3p21, 3p22-24, 3p25 and 9p21; (4) The size of the 3p deletions increased with progressive histologic changes; (5) TP53 allelic loss was present in many histologically advanced lesions (dysplasia and CIS); (6) Analyses of 58 normal and non-invasive foci having any molecular abnormality, indicated that 30 probably arose as independent clonal events, while 28 were potentially of the same clonal origin as the corresponding tumor; (7) Nevertheless, when the allelic losses in the 30 clonally independent lesions and their clonally unrelated tumors were compared the same parental allele was lost in 113 of 125 (90%) of comparisons. The mechanism by which this phenomenon (known as allele speci®c mutations) occurs is unknown; (8) Four patterns of allelic loss in clones were found. Histologically normal or mildly abnormal foci had a negative pattern (no allelic loss) or early pattern of loss while all foci of CIS and invasive tumor had an advanced pattern. However dysplasias demonstrated the entire spectrum of allelic loss patterns, and were the only histologic category having the intermediate pattern. Our ®ndings indicate that multiple, sequentially occurring allele speci®c molecular changes commence in widely dispersed, apparently clonally independent foci, early in the multistage pathogenesis of squamous cell carcinomas of the lung.

show abstract

Section: Polymorphic Dna Markers and Pcr-loh Analysismentioning

confidence: 99%

Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma

et al. 1999

View full text Add to dashboard Cite

show abstract

“…A "touchdown" procedure [14] was used with a 18C decrement of the annealing temperature for the first 10 cycles. A total of 50 cycles were performed.…”

Section: Chlamydia Pneumoniae Polymerase Chain Reactionmentioning

confidence: 99%

Chlamydia pneumoniae infection in adults with chronic cough compared with healthy blood donors

Birkebæk¹,

Jensen

Seefeldt

et al. 2000

Eur Respir J

View full text Add to dashboard Cite

In a small uncontrolled study, persistent cough has recently been found to be associated with serological evidence of acute Chlamydia pneumoniae infection. In order to assess whether C. pneumoniae plays a role in chronic cough, the prevalence of C. pneumoniae infection in 201 adult patients with chronic cough was compared with the prevalence in 106 healthy blood donors without respiratory tract symptoms in the preceding 3 months. A microimmunofluorescence antibody test was used to determine C. pneumoniae antibodies in the immunoglobulin (Ig)M, IgG and IgA fractions. Further, nasopharyngeal aspirates from the 201 patients were examined for C. pneumoniae deoxyribonucleic acid by polymerase chain reaction (PCR).As judged by serology, nine patients (4%) and one control (1%) had acute C. pneumoniae infection, and 92 patients (46%) and 42 controls (40%) had previous or chronic C. pneumoniae infection. Of the nine patients with acute infection, three were C. pneumoniae PCR positive, and they all had an IgM antibody titre response. The remaining six patients had either an IgG antibody titre of $512 (five patients) or an IgA antibody titre of $512 (one patient). None of these six patients had detectable IgM antibodies.The mean cough period for the five IgG positive patients (10.8 weeks) was significantly longer than the mean cough period for the remaining patient population (6.4 weeks; p=0.004).It is concluded that Chlamydia pneumoniae infection was not statistically significantly more prevalent in patients with chronic cough than in healthy blood donors, and that Chlamydia pneumoniae appears to have a minor role in patients with chronic cough. Direct detection of Chlamydia pneumoniae by polymerase chain reaction on nasopharyngeal aspirates is highly correlated with detectable immunoglobulin M antibodies, but in the late stages of prolonged cough serological testing of immunoglobulin G and immunoglobulin A may be more beneficial for obtaining a microbiological diagnosis. Eur Respir J 2000; 16: 108±111.

show abstract

“…Using Touchdown-PCR (Don et al, 1991), with primers designed from published mammalian CDKN2 gene family sequences, a 130 bp amplimer derived from X. maculatus genomic DNA was initially obtained (Nairn et al, 1996b). Computer translation of the DNA sequence revealed it was probably representative of a ®sh CDKN2 gene family member.…”

Section: Cdkn2x Cloning and Structural Organizationmentioning

confidence: 99%

“…Using Touchdown-PCR (Don et al, 1991) with primers designed based on published CDKN2 gene family sequences, a 130 bp amplimer derived from the genome of X. maculatus was initially cloned and sequenced (Nairn et al, 1996b). Oligonucleotide primers were then designed based on sequence information from this clone, for use in`Inverse' -PCR (Ochman et al, 1988) using X. helleri EcoRI-digested DNA.…”

Section: Cloning and Sequencing Of Cdkn2xmentioning

confidence: 99%

Comparative structure and characterization of a CDKN2 gene in a Xiphophorus fish melanoma model

Kazianis

Coletta

et al. 1999

Oncogene

View full text Add to dashboard Cite

We have cloned, sequenced, and characterized the RNA expression properties of a ®sh CDKN2 gene from Xiphophorus helleri and X. maculatus. This gene, termed CDKN2X, shows a high degree of amino acid sequence similarity to members of the mammalian CDKN2 gene family, which includes the tumor suppressor loci CDKN2A (P16) and CDKN2B (P15). Comparative sequence analysis suggests that ®sh CDKN2X is similarly related to all four mammalian gene family members, and may represent a descendant of an ancestral prototypic CDKN2 gene. CDKN2X was mapped to a region on autosomal Xiphophorus linkage group V (LG V) known to contain the DIFF gene that acts as a tumor suppressor of melanoma formation in X. helleri/X. maculatus backcross hybrids. Thus, CDKN2X may be a candidate for the tumor suppressor DIFF gene. Here we have sequenced CDKN2X in both Xiphophorus species and have characterized its expression in normal and melanotic tissues within control and backcross hybrid ®sh. A simultaneous expressional analysis of the Xmrk-2 tyrosine kinase receptor gene, which is strongly implicated in melanomagenesis in this system, was also performed. RT ± PCR analyses revealed that both genes were highly expressed in melanomas. For CDKN2X, this result contrasts numerous ®ndings in human tumors including human melanoma in which either CDKN2A (P16) deactivation or LOH was observed.

show abstract

‘Touchdown’ PCR to circumvent spurious priming during gene amplification

Cited by 2,402 publications

References 4 publications

Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma

Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma

Chlamydia pneumoniae infection in adults with chronic cough compared with healthy blood donors

Comparative structure and characterization of a CDKN2 gene in a Xiphophorus fish melanoma model

Contact Info

Product

Resources

About