Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (89K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (175)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Liu, W.
Right arrow Articles by James, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, W.
Right arrow Articles by James, C. D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research Pages 1396-1400

Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations
Introduction
Materials And Methods
   Amplicon synthesis
   Denaturing HPLC analysis
   Sequence analysis
   Microsatellite analysis
Results And Discussion
Acknowledgements
References


Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations

Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations Wanguo Liu, David I. Smith, Keri J. Rechtzigel, Stephen N. Thibodeau and C. David James*

Department of Laboratory Medicine and Pathology, Division of Experimental Pathology, Mayo Clinic and Foundation, Rochester, MN 55905, USA

Received December 17, 1997; Revised and Accepted January 27, 1998

ABSTRACT

Denaturing high performance liquid chromatography (DHPLC) has been described recently as a method for screening DNA samples for single nucleotide polymorphisms and inherited mutations. Thirty-eight DNAs, 22 of which were heterozygous for previously characterized rearranged transforming gene (RET) or cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations or polymorphisms, were examined using DHPLC analysis to assess the accuracy of this scanning method. Ninety-one per cent (20/22) of the PCR amplicons from specimens with heterozygous RET or CFTR sequence showed elution profiles distinct from corresponding homozygous normal patterns; whether the profiles for two amplicons containing heterozygous RET sequence were distinct from homozygous cases was equivocal. To investigate the usefulness of this method for detecting mutations in tumor DNAs, each of the phosphatase and tensin homologue deleted on chromosome ten gene (PTEN) exons were examined for mutations in 63 malignant gliomas. Seventeen PTEN PCR products from this series of brain tumors showed elution profiles indicating sample heterozygosity and in each instance conventional sequencing confirmed the presence of a mutation. PTEN amplicons containing exons 1, 3 and 5 were sequenced for each of the 63 tumor DNAs to determine whether any mutations may have escaped DHPLC detection, and this analysis identified one such alteration in addition to the eight mutations that DHPLC had revealed. In total, DHPLC identified 37 of 40 (92.5%) PCR products containing defined sequence variation and no alterations were indicated among 196 amplicons containing homozygous normal sequence.

INTRODUCTION

It is of fundamental importance to both basic and clinical research to efficiently and accurately detect gene sequence variation within DNA samples. Several methods have been developed to scan DNAs for polymorphisms and mutations to accommodate this need, and these techniques have been reviewed on multiple occasions (1-4).

A relatively new addition to DNA scanning methods uses denaturing high performance liquid chromatography (DHPLC; 5-9). In its early stage of application to the analysis of nucleic acids, HPLC was shown to provide an effective means for separating oligonucleotides (10), PCR fragments (11) and for analyzing the products formed in competitive RT-PCR reactions to determine relative levels of gene expression (12).

Mutation/polymorphism scanning by DHPLC involves subjecting PCR products to ion-pair reverse-phase liquid chromatography in a column containing alkylated non-porous particles. Under conditions of partial heat denaturation within a linear acetonitrile gradient, heteroduplexes that form in PCR samples having internal sequence variation display reduced column retention time relative to their homoduplex counterparts. In the majority of cases the elution profiles for such samples are distinct from those having homozygous sequence, making the identification of samples harboring polymorphisms or mutations a straightforward procedure. The major advantages of this method include the use of automated instrumentation, speed of analysis (~5 min per sample) and the size of the DNA fragment that can be analyzed (up to 1.5 kb).

No previous report has addressed the accuracy of mutation/polymorphism detection by DHPLC analysis. One of the objectives of the investigation reported here was to determine the reliability of DHPLC for detecting inherited gene sequence variation. To accomplish this we used DHPLC to examine PCR fragments produced from several DNAs, having previously identified germline mutations or polymorphisms in the rearranged transforming proto-oncogene (RET) or the cystic fibrosis transmembrane conductance regulator gene (CFTR). Our other major interest was to assess the usefulness of DHPLC for screening tumor DNAs for mutations of tumor suppressor genes (TSGs), a potentially powerful application of this technology that had not previously been examined. However, as the method requires heteroduplex DNA for detection of intra-sample sequence variation, it is reasonable to question whether mutations would escape detection in instances where loss of a wild-type TSG occurs in combination with mutation of the remaining allele since the predominant double-stranded DNA formed would be mutant homoduplex. To address this question, a large panel of malignant glioma DNAs were examined for phosphatase and tensin homologue deleted on chromosome ten gene (PTEN) mutations. The results of these analyses indicate that DHPLC offers a reliable approach for the detection of germline and somatic mutations.

MATERIALS AND METHODS

Amplicon synthesis

DNAs from peripheral blood leukocytes and tumor tissue snap frozen by immersion in liquid nitrogen were isolated and purified as described (13). Samples used for mutation screening and sequencing were generated in 50 µl reaction volumes containing 10-100 ng of genomic DNA, 20 pmol of forward and reverse primers for either PTEN exons 1-9 (14), CFTR exon 7 (15) or RET exon 10 (16), 200 µM dNTPs (Perkin-Elmer, Foster City, CA), 1.25 U of Taq polymerase (AmpliTaq Gold: Perkin-Elmer) and 1× buffer supplied by the manufacturer. PCR amplifications were for 35 cycles: 95°C for 30 s, 60°C for 30 s and 72°C for 1 min (final extension at 72°C for 10 min) following sample denaturation at 95°C for 9 min. Synthesis of appropriately sized PCR reaction products was confirmed by agarose gel electrophoresis.

Denaturing HPLC analysis

DHPLC analysis was carried out using automated instrumentation identical to that described by Underhill et al. (9). Four to seven µl of each PCR product, containing ~50-100 ng DNA, was denatured for 3 min at 95°C and then gradually reannealed by decreasing sample temperature from 95 to 65°C over a period of 30 min. PCR products were then separated (flow rate of 0.9 ml/min) over a period of time and through a linear acetonitrile gradient, the values for which were determined by the size and G-C content of the amplicon (Table 1).


Table 1. Comparison of mutation detection by DHPLC and by sequencing

1Mobile phase temperature.
2cDNA sequence location of alteration in parentheses.3HT, heterozygous; HM, homozygous.
Italicized type indicates cases in which the results of DHPLC and conventional sequencing were discrepant. Altering the mobile phase temperature in these instances, however, resolved sample homoduplex and heteroduplex fractions (see Results and Discussion).

The column mobile phase consisted of a mixture of 0.1 M triethylamine acetate (pH 7.0) with (buffer A) or without (buffer B) 25% acetonitrile. The mobile phase temperatures required for optimal resolution of homoduplex and heteroduplex DNAs were determined empirically by injecting one PCR product for each exon at increasing temperatures until a significant decrease in sample retention time was observed. Specific values for the gradient ranges (buffer A component indicated), separation times and mobile phase temperatures used to analyze the amplicons described above are as follows: 57.0-64.2%, 4 min and 58°C for CFTR exon 7; 53.0-59.3%, 3.5 min and 61°C or 53.0-59.3%, 3.5 min and 59°C for RET exon 10; 55.2-56.2%, 5 min and 59°C for PTEN exon 1; 52.0-57.4%, 3 min and 58°C for PTEN exon 3 and 54.5-60.8%, 3.5 min and 57°C for PTEN exon 5. Between sample analyses the column was regenerated with a 19:1 mixture of buffers A and B (40 s) and a solution whose buffer A content was 5% less than the low end of the desired gradient range (40 s).

Sequence analysis

Solutions (10 µl) were prepared with 10-20 ng of product from previous PCR reactions, 0.05 U of Taq polymerase, 1× buffer, 10% DMSO, 400 µM ddATP, 600 µM ddTTP, 60 µM ddGTP, 200 µM ddCTP, 10 µM each of dATP, dTTP and dCTP, 20 µM 7-deaza-dGTP (Boehringer Mannheim) and 0.05 µM 5'-32P-labeled sequencing primer. Sequencing reactions were carried out for 30 cycles at 95°C for 20 s, 58°C for 30 s and 72°C for 1 min, using a 1 min ramp time between annealing and elongation phases. Following sample denaturation, reaction products were loaded onto a 6% sequencing gel. Electrophoresis was at 75 W and room temperature for 1-3 h, after which the gels were dried and exposed to Kodak XAR film.

Microsatellite analysis

PCR reactions for determination of tumor loss of heterozygosity contained ~10 ng of genomic DNA, 8-10 pM forward and reverse primers for either the D10S541 or D10S1765 locus (Research Genetics, Huntsville, AL), 0.8 µCi [[alpha]-32P]dCTP and 0.2-0.35 U of Taq polymerase in 10-15 µl of 1× buffer containing 200 µM dGTP, dATP and dTTP, and 25-34 µM dCTP. Samples were placed in 96-well plates and amplified at 95°C denaturation (30 s), 55°C annealing (30 s) and 72°C extension (1 min) for 43 cycles. At completion of PCR, an equal volume of denaturing buffer was added to each reaction. Samples were then heated to 95°C and quenched on ice. Two µl of each sample were applied to 4 or 6% acrylamide sequencing gels and electrophoresed for 1.5-3 h at 75 W. Gels were dried and exposed to X-ray film for 4-48 h.


Figure 1. DHPLC detection of CFTR and RET germline mutations. Elution profiles associated with the DHPLC analysis of PCR amplicons containing either CFTR exon 7 (A) or RET exon 10 (B). Retention times for homoduplex and heteroduplex fractions are indicated above the CFTR elution peaks. For the RET exon 10 results, only the portion of the profile containing the homoduplex and heteroduplex peaks are shown. cDNA sequence location of mutations identified previously in each sample are indicated. The inset profile shown for the T -> C substitution at base 1852 was obtained using a mobile phase temperature of 59°C.

RESULTS AND DISCUSSION

PCR fragments were synthesized from 22 peripheral blood leukocyte specimens heterozygous for previously identified exon 10 RET or exon 7 CFTR mutations or polymorphisms (Table 1). Each PCR reaction product was subjected to DHPLC analysis and their corresponding elution profiles were compared with patterns associated with homozygous normal sequence controls, nine of which were included for the analysis of CFTR sequence alterations and seven for the analysis of RET alterations.

The elution profiles for the control CFTR PCR products were all highly similar and showed a single peak of homoduplex DNA. In contrast, each of the nine PCR products with internal CFTR sequence variation produced a distinct profile with multiple peaks due to the reduced column retention time of heteroduplex DNA (examples shown in Fig. 1A). All samples with heterozygous CFTR sequence were identified using the same separation conditions (Materials and Methods). G-C content of the 60 bases surrounding each CFTR alteration varied between 35 and 54%, suggesting that the detection of sample heteroduplex within a specific amplicon is not greatly influenced by differences in the melting point of sequences flanking the site of base mismatch.

To determine whether DHPLC detection of sample heterozygosity is influenced by nucleotide identity at a specific site of sequence variation, several patient DNAs with heterozygous mutations effecting RET cysteine codons 609, 611, 618 and 620 were examined. For nucleotide substitutions at position 1852 of the coding sequence, split-peak elution profiles distinct from the profiles associated with normal homoduplex DNAs were evident for T -> A and T -> G alterations (Fig. 1B). However, a T -> C substitution at this position failed to produce a profile with multiple peaks; this was also the case for an amplicon containing a G -> A alteration at base 1859 (Table 1). The peaks for these two cases, however, were noticeably wider than control peaks, and thereby suggested the presence of homoduplexes and heteroduplexes in the corresponding eluates. An alternative DHPLC protocol (mobile phase temperature of 59°C) resulted in a slight resolution of homoduplex and heteroduplex fractions in each sample (see inset for the T -> C substitution at base 1852, Fig. 1B). Nine additional heterozygous samples with mutations effecting the cysteine codons showed unique profiles using the initial separation protocol, including two associated with different nucleotide substitutions at position 1853 (Fig. 1B). Significantly, there were no false positives associated with the analysis of either CF or RET sequence alterations.


Figure 2. DHPLC detection of PTEN mutations in tumor DNAs. Portions of elution profiles are shown for homoduplex and heteroduplex peaks resulting from the analysis of normal or tumor PTEN exon 5 PCR products (A) and for PTEN exon 7 PCR products synthesized from a mixed sample containing homozygous normal DNA and DNA from a glioblastoma cell line with a PTEN exon 7 mutation (B). Locations of corresponding exon 5 mutations are shown with the profiles to the left (A) and corresponding proportions of cell line:normal DNA are indicated for the profiles shown to the right (B).

Mutation screening by DHPLC has not been applied to the analysis of DNA samples extracted from neoplastic tissue, and to assess the potential of this technology for analyzing tumor specimens, we examined DNAs from 63 malignant gliomas for mutation of the PTEN gene. Elution profiles indicated sequence variation within 17 of the 567 PCR products examined, and in each of these cases the presence of a mutation was determined by conventional sequencing (elution profiles for DNAs with exon 5 mutations are shown in Fig. 2A). As opposed to the RET and CFTR germline mutations which involved single nucleotide substitutions in all instances, the PTEN mutations included deletions and insertions, as well as several nucleotide substitutions (Table 1). The overall incidence of DHPLC-detected PTEN mutations among this series of tumors (17 of 63; 27%) compares favorably with those reported previously (14,17) and, as was the case for the analysis of DNAs for germline CFTR or RET mutations, there were no false positives associated with the PTEN analysis.

To determine whether some alterations had escaped DHPLC detection, PTEN PCR fragments for exons 1, 3 and 5 were conventionally sequenced for all 63 cases. This analysis revealed a single exon 3 mutation that DHPLC had failed to identify. Interestingly, the exon 3 PCR product had the lowest G-C composition (27%) of any of the amplicons examined in this study. Comparison of mobile phase temperatures (MPTs) against corresponding amplicon G-C contents in Table 1 suggests that the temperature used to analyze exon 3 amplicons may have been too high and prevented mutation detection by `melting open' sample duplexes. Consequently, we compared our empirically derived MPTs (Materials and Methods) against MPTs recommended by a recently installed internet program (http://lotka.stanford.edu/dhplc/melt.html ). This analysis revealed a close correspondence between experimental and recommended MPTs for all amplicons other than exon 3, for which the internet program recommended a temperature of 53°C. DHPLC analysis at this temperature clearly revealed homoduplex and heteroduplex fractions in the tumor specimen containing the exon 3 mutation (data not shown).

PTEN mutations in malignant gliomas are often accompanied by loss of the remaining wild-type allele (14,17,18). To assess whether this had occurred in any of the tumors examined here, microsatellite analysis was performed on 14 samples with PTEN mutations for which there was corresponding normal DNA available. This analysis revealed loss of heterozygosity in 13 instances and, consequently, these results suggest that DHPLC can detect the formation of heteroduplex even when the ratio of normal:mutant DNA sequence in a tumor DNA is quite low. To formally test this hypothesis, we mixed varying amounts of DNA from normal tissue and cell line U251, homozygous for a dinucleotide insertion mutation in PTEN exon 7 (14), and analyzed resulting exon 7 PCR amplicons. Elution profiles from the DHPLC analysis of these samples indicate that substantial heteroduplex is formed even in instances where the tumor DNA is 4-fold more abundant than normal (Fig. 2B); similar results were obtained for the reverse situation where normal DNA represented the majority component of the mixture. Taken together, these results indicate that DHPLC requires between 10 and 20% of the minority DNA species for detecting heteroduplex DNA, and extend the use of this method to tumor mutational analysis. In addition, this experiment shows that DHPLC can be used to detect alterations in a homogenous mutant DNA (e.g. cell line) sample by adding an approximately equal amount of normal DNA to the clonal, mutant specimen.

In summary, this survey of different exon sequences indicates that DHPLC offers a reliable and sensitive means for the detection of germline and somatic mutations. The few exceptions encountered may relate to the extreme G-C content of the associated amplicons (64 and 27% for RET exon 10 and PTEN exon 3, respectively). The differential sensitivity for detecting the transversion and transition mismatches at RET positions 1852 and 1853 are surprising, but imply that DHPLC profiles may serve as a type of `fingerprint' revealing the precise sequence alteration associated with sample heterogeneity. At a minimum, the accuracy of the method suggests a potential for increased use.

ACKNOWLEDGEMENTS

This work was supported by NCI grants CA-55728 (C.D.J.) and CA-48031 (D.I.S.).

REFERENCES

1. Grompe, M. (1993) Nature Genet., 5, 111-117. MEDLINE Abstract

2. Forrest, S., Cotton, R., Landegren, U. and Southern, E. (1995) Nature Genet., 10, 375-376. MEDLINE Abstract

3. Mashal, R.D. and Sklar, J. (1996) Curr. Opin. Genet. Dev., 6, 275-280. MEDLINE Abstract

4. Cotton, R.G.H. (1997) Trends Genet., 13, 43-66.

5. Oefner, P.J. and Underhill, P.A. (1995) Am. J. Hum. Genet., 57 (Suppl.), A266.

6. Underhill, P.A., Jin, L., Zemans, R., Oefner, P.J. and Cavalli-Sforza, L.L. (1996) Proc. Natl. Acad. Sci. USA, 93, 196-200. MEDLINE Abstract

7. Ophoff, R.A., Terwindt, G.M., Vergouwe, M.N., van Eijk, R., Oefner, P.J., Hoffman, S.M., Lamerdin, J.E., Mohrenweiser, H.W., Bulman, D.E., Ferrari, M., Haan, J., Lindhout, D., van Ommen, G.J., Hofker, M.H., Ferrari, M.D. and Frants, R.R. (1996) Cell, 87, 543-552. MEDLINE Abstract

8. Hayward-Lester, A., Chilton, B.S., Underhill, P.A., Oefner, P.J. and Doris, P.S. (1996) In Ferre, F. (ed.), Gene Quantification. Birkhauser Verlag, Basel Switzerland, pp. 44-77.

9. Underhill, P.A., Jin, L., Lin, A.A., Mehdi, S.Q., Jenkins, T., Vollrath, D., Davis, R.W., Cavalli-Sforza, L.L. and Oefner, P.J. (1997) Genome Res., 7, 996-1005. MEDLINE Abstract

10. Huber, C.G., Oefner, P.J. and Bonn, G.K. (1993) Anal. Biochem., 212, 351-358. MEDLINE Abstract

11. Huber, C.G., Oefner, P.J., Preuss, E. and Bonn, G.K. (1993) Nucleic Acids Res., 21, 1061-1066. MEDLINE Abstract

12. Hayward-Lester, A., Oefner, P.J., Sabatini, S. and Doris, P.A. (1995) Genome Res., 5, 494-499. MEDLINE Abstract

13. James, C.D., Carlbom, E., Dumanski, J., Hansen, M., Nordenskjold, M., Collins, V.P. and Cavenee, W.K. (1988) Cancer Res., 48, 5546-5551. MEDLINE Abstract

14. Steck, P.A., Pershouse, M.A., Jasser, S.A., Yung, W.K.A, Lin, H., Lignon, A.H., Langford, L.A., Baumgard, M.L., Hattier, T., Davis, T., Frye, C., Hu, R., Swedlund, B., Teng, D.H.F. and Tavtigian, S.V. (1997) Nature Genet., 15, 356-362. MEDLINE Abstract

15. Zielenski, J., Rozmahel, R., Bozon, D., Kerem, B., Grzelczak, Z., Riordan, J.R., Rommens, J. and Tsui, L.C. (1991) Genomics, 10, 214-228. MEDLINE Abstract

16. Tsai, M.S., Ledger, G.A., Khosla, S., Gharib, H. and Thibodeau, S.N. (1994) J. Clin. Endocrinol. Metab., 78, 1261-1264. MEDLINE Abstract

17. Rasheed, B.K.A., Stenzel, T.T., Mclendon, R.E., Parsons, R., Friedman, A.H., Friedman, H.S., Bigner, D.D. and Bigner, S.H. (1997) Cancer Res., 57, 4187-4190. MEDLINE Abstract

18. Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S.I., Janusz, P., Miliaresis, C., Rodgers, L., McCombie, R., Bigner, S.H., Giovanella, B.C., Ittmann, M., Tycko, B., Hibshoosh, H., Wigler, M.H. and Parsons, R. (1997) Science, 275, 1943-1947. MEDLINE Abstract

*To whom correspondence should be addressed at: Mayo Clinic and Foundation, 200 First Street S.W., Hilton Building Room 820-D, Rochester, MN 55905, USA. Tel: +1 507 284 8989; Fax: +1 507 266 5193; Email: james.charles@mayo.edu

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 27 Feb 1998
Copyright© Oxford University Press, 1998.

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Appl. Environ. Microbiol.Home page
A. O. Wagner, C. Malin, and P. Illmer
Application of Denaturing High-Performance Liquid Chromatography in Microbial Ecology: Fermentor Sludge, Compost, and Soil Community Profiling
Appl. Envir. Microbiol., February 15, 2009; 75(4): 956 - 964.
[Abstract] [Full Text] [PDF]

Home page
 J Biomol ScreenHome page
A. Piepoli, E. Schirru, A. Mastrorilli, A. Gentile, R. Cotugno, M. Quitadamo, A. Merla, M. Congia, P. U. Satta, and F. Perri
Genotyping of the Lactase-Phlorizin Hydrolase C/T-13910 Polymorphism by Means of a New Rapid Denaturing High-Performance Liquid Chromatography-Based Assay in Healthy Subjects and Colorectal Cancer Patients
J Biomol Screen, August 1, 2007; 12(5): 733 - 739.
[Abstract] [PDF]

Home page
 Cancer Res.Home page
X. Wang, F. Wang, K. Taniguchi, R. S. Seelan, L. Wang, K. E. Zarfas, S. K. McDonnell, C. Qian, K. Pan, Y. Lu, et al.
Truncating Variants in p53AIP1 Disrupting DNA Damage-Induced Apoptosis Are Associated with Prostate Cancer Risk
Cancer Res., November 1, 2006; 66(21): 10302 - 10307.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
G. Amicarelli, D. Adlerstein, E. Shehi, F. Wang, and G. M. Makrigiorgos
Genotype-Specific Signal Generation Based on Digestion of 3-Way DNA Junctions: Application to KRAS Variation Detection
Clin. Chem., October 1, 2006; 52(10): 1855 - 1863.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
S. Matsukuma, M. Yoshihara, F. Kasai, A. Kato, A. Yoshida, M. Akaike, O. Kobayashi, H. Nakayama, Y. Sakuma, T. Yoshida, et al.
Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay
J. Mol. Diagn., September 1, 2006; 8(4): 504 - 512.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
M. Crepin, P. Pigny, F. Escande, C. C. Bauters, A. Calender, S. Lefevre, M.-P. Buisine, N. Porchet, and M.-F. Odou
Evaluation of denaturing high performance liquid chromatography for the mutational analysis of the MEN1 gene.
J. Mol. Endocrinol., April 1, 2006; 36(2): 369 - 376.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
J. Li, L. Harris, H. Mamon, M. H. Kulke, W.-H. Liu, P. Zhu, and G. Mike Makrigiorgos
Whole Genome Amplification of Plasma-Circulating DNA Enables Expanded Screening for Allelic Imbalance in Plasma
J. Mol. Diagn., February 1, 2006; 8(1): 22 - 30.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
K Laud, C Marian, M F Avril, M Barrois, A Chompret, A M Goldstein, M A Tucker, P A Clark, G Peters, V Chaudru, et al.
Comprehensive analysis of CDKN2A (p16INK4A/p14ARF) and CDKN2B genes in 53 melanoma index cases considered to be at heightened risk of melanoma
J. Med. Genet., January 1, 2006; 43(1): 39 - 47.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
J. Bacani, R. Zwingerman, N. Di Nicola, S. Spencer, T. Wegrynowski, K. Mitchell, K. Hay, M. Redston, E. Holowaty, D. Huntsman, et al.
Tumor Microsatellite Instability in Early Onset Gastric Cancer
J. Mol. Diagn., October 1, 2005; 7(4): 465 - 477.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
M. Hegde, M. Blazo, B. Chong, T. Prior, and C. Richards
Assay Validation for Identification of Hereditary Nonpolyposis Colon Cancer-Causing Mutations in Mismatch Repair Genes MLH1, MSH2, and MSH6
J. Mol. Diagn., October 1, 2005; 7(4): 525 - 534.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Pathol.Home page
B Yu, N A Sawyer, M Caramins, Z G Yuan, R B Saunderson, R Pamphlett, D R Richmond, R W Jeremy, and R J Trent
Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease
J. Clin. Pathol., May 1, 2005; 58(5): 479 - 485.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
L.-S. Chou, F. Gedge, and E. Lyon
Complete Gene Scanning by Temperature Gradient Capillary Electrophoresis Using the Cystic Fibrosis Transmembrane Conductance Regulator Gene as a Model
J. Mol. Diagn., February 1, 2005; 7(1): 111 - 120.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
P. Yang, W.R. Bamlet, J.O. Ebbert, W.R. Taylor, and M. de Andrade
Glutathione pathway genes and lung cancer risk in young and old populations
Carcinogenesis, October 1, 2004; 25(10): 1935 - 1944.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
D. P. Steensma, D. R. Higgs, C. A. Fisher, and R. J. Gibbons
Acquired somatic ATRX mutations in myelodysplastic syndrome associated with {alpha} thalassemia (ATMDS) convey a more severe hematologic phenotype than germline ATRX mutations
Blood, March 15, 2004; 103(6): 2019 - 2026.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. J. Fuja, F. Lin, K. E. Osann, and P. J. Bryant
Somatic Mutations and Altered Expression of the Candidate Tumor Suppressors CSNK1{epsilon}, DLG1, and EDD/hHYD in Mammary Ductal Carcinoma
Cancer Res., February 1, 2004; 64(3): 942 - 951.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
E. Esteban-Cardenosa, M. Duran, M. Infante, E. Velasco, and C. Miner
High-Throughput Mutation Detection Method to Scan BRCA1 and BRCA2 Based on Heteroduplex Analysis by Capillary Array Electrophoresis
Clin. Chem., February 1, 2004; 50(2): 313 - 320.
[Abstract] [Full Text] [PDF]

Home page
 NeurologyHome page
F. Sicca, A. Kelemen, P. Genton, S. Das, D. Mei, F. Moro, W.B. Dobyns, and R. Guerrini
Mosaic mutations of the LIS1 gene cause subcortical band heterotopia
Neurology, October 28, 2003; 61(8): 1042 - 1046.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
M. Bianchini, E. Ottaviani, T. Grafone, B. Giannini, S. Soverini, C. Terragna, M. Amabile, P. P. Piccaluga, M. Malagola, M. Rondoni, et al.
Rapid Detection of Flt3 Mutations in Acute Myeloid Leukemia Patients by Denaturing HPLC
Clin. Chem., October 1, 2003; 49(10): 1642 - 1650.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
L. Zhang, W. Lu, X. Miao, D. Xing, W. Tan, and D. Lin
Inactivation of DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation and its relation to p53 mutations in esophageal squamous cell carcinoma
Carcinogenesis, June 1, 2003; 24(6): 1039 - 1044.
[Abstract] [Full Text] [PDF]

Home page
 Clin Med ResHome page
R. J. Wurzburger, R. Gupta, A. P. Parnassa, S. Jain, J. A. Wexler, J. L. Chu, K. B. Elkon, and R. D. Blank
Use of GC Clamps in DHPLC Mutation Scanning
Clin. Med. Res., April 1, 2003; 1(2): 111 - 118.
[Abstract] [Full Text] [PDF]

Home page
 J Antimicrob ChemotherHome page
F. Hannachi-M'Zali, J. E. Ambler, C. F. Taylor, and P. M. Hawkey
Examination of single and multiple mutations involved in resistance to quinolones in Staphylococcus aureus by a combination of PCR and denaturing high-performance liquid chromatography (DHPLC)
J. Antimicrob. Chemother., November 1, 2002; 50(5): 649 - 655.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
A. Gallo, E. Thomson, J. Brindle, M. A. O'Connell, and L. P. Keegan
Micro-processing events in mRNAs identified by DHPLC analysis
Nucleic Acids Res., September 15, 2002; 30(18): 3945 - 3953.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Nakagawa, H. Yan, J. Lockman, H. Hampel, K. W. Kinzler, B. Vogelstein, and A. de la Chapelle
Allele Separation Facilitates Interpretation of Potential Splicing Alterations and Genomic Rearrangements
Cancer Res., August 15, 2002; 62(16): 4579 - 4582.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
D J Halliday, S Hutchinson, L Lonie, J A Hurst, H Firth, P A Handford, and P Wordsworth
Twelve novel FBN1 mutations in Marfan syndrome and Marfan related phenotypes test the feasibility of FBN1 mutation testing in clinical practice
J. Med. Genet., August 1, 2002; 39(8): 589 - 593.
[Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
M.G. Katz, B. Chu, R. McLachlan, N.I. Alexopoulos, D.M. de Kretser, and D.S. Cram
Genetic follow-up of male offspring born by ICSI, using a multiplex fluorescent PCR-based test for Yq deletions
Mol. Hum. Reprod., June 1, 2002; 8(6): 589 - 595.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
D Morrison, D FitzPatrick, I Hanson, K Williamson, V van Heyningen, B Fleck, I Jones, J Chalmers, and H Campbell
National study of microphthalmia, anophthalmia, and coloboma (MAC) in Scotland: investigation of genetic aetiology
J. Med. Genet., January 1, 2002; 39(1): 16 - 22.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
M. Zariwala, P. G. Noone, A. Sannuti, S. Minnix, Z. Zhou, M. W. Leigh, M. Hazucha, J. L. Carson, and M. R. Knowles
Germline Mutations in an Intermediate Chain Dynein Cause Primary Ciliary Dyskinesia
Am. J. Respir. Cell Mol. Biol., November 1, 2001; 25(5): 577 - 583.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
I. Orlow, P. Roy, A. Barz, R. Canchola, Y. Song, and M. Berwick
Validation of Denaturing High Performance Liquid Chromatography as a Rapid Detection Method for the Identification of Human INK4A Gene Mutations
J. Mol. Diagn., November 1, 2001; 3(4): 158 - 163.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. B. Lee, S. H. Kim, D. W. Bell, D. C. R. Wahrer, T. A. Schiripo, M. M. Jorczak, D. C. Sgroi, J. E. Garber, F. P. Li, K. E. Nichols, et al.
Destabilization of CHK2 by a Missense Mutation Associated with Li-Fraumeni Syndrome
Cancer Res., November 1, 2001; 61(22): 8062 - 8067.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
U. zur Stadt, J. Rischewski, R. Schneppenheim, and H. Kabisch
Denaturing HPLC for Identification of Clonal T-Cell Receptor {gamma} Rearrangements in Newly Diagnosed Acute Lymphoblastic Leukemia
Clin. Chem., November 1, 2001; 47(11): 2003 - 2011.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
G. Le Gac, C. Mura, and C. Ferec
Complete Scanning of the Hereditary Hemochromatosis Gene (HFE) by Use of Denaturing HPLC
Clin. Chem., September 1, 2001; 47(9): 1633 - 1640.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
S. Rickard, D. P Kelsell, T. Sirimana, K. Rajput, B. MacArdle, and M. Bitner-Glindzicz
Recurrent mutations in the deafness gene GJB2 (connexin 26) in British Asian families
J. Med. Genet., August 1, 2001; 38(8): 530 - 533.
[Full Text] [PDF]

Home page
 IOVSHome page
H. P. N. Scholl, J. Kremers, R. Vonthein, K. White, and B. H. F. Weber
L- and M-Cone-Driven Electroretinograms in Stargardt's Macular Dystrophy-Fundus Flavimaculatus
Invest. Ophthalmol. Vis. Sci., May 1, 2001; 42(6): 1380 - 1389.
[Abstract] [Full Text]

Home page
 Clin. Chem.Home page
E. Schaeffeler, T. Lang, U. M. Zanger, M. Eichelbaum, and M. Schwab
High-Throughput Genotyping of Thiopurine S-Methyltransferase by Denaturing HPLC
Clin. Chem., March 1, 2001; 47(3): 548 - 555.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
B. J. C. van den Bosch, R. F. M. de Coo, H. R. Scholte, J. G. Nijland, R. van den Bogaard, M. de Visser, C. E. M. de Die-Smulders, and H. J. M. Smeets
Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography
Nucleic Acids Res., October 15, 2000; 28(20): e89 - e89.
[Abstract] [Full Text] [PDF]

Home page
 Genome ResHome page
H. Tian, L. C. Brody, and J. P. Landers
Rapid Detection of Deletion, Insertion, and Substitution Mutations via Heteroduplex Analysis Using Capillary- and Microchip-Based Electrophoresis
Genome Res., September 1, 2000; 10(9): 1403 - 1413.
[Abstract] [Full Text]

Home page
 Clin. Chem.Home page
D. Pirulli, M. Giordano, D. Puzzer, S. Crovella, I. Rigato, C. Tiribelli, P. Momigliano-Richiardi, and A. Amoroso
Rapid Method for Detection of Extra (TA) in the Promoter of the Bilirubin-UDP-Glucuronosyl Transferase 1 Gene Associated with Gilbert Syndrome
Clin. Chem., January 1, 2000; 46(1): 129 - 131.
[Full Text] [PDF]

Home page
 ScienceHome page
D. W. Bell, J. M. Varley, T. E. Szydlo, D. H. Kang, D. C. Wahrer, K. E. Shannon, M. Lubratovich, S. J. Verselis, K. J. Isselbacher, J. F. Fraumeni, et al.
Heterozygous Germ Line hCHK2 Mutations in Li-Fraumeni Syndrome
Science, December 24, 1999; 286(5449): 2528 - 2531.
[Abstract] [Full Text]

Home page
 Proc. Natl. Acad. Sci. USAHome page
W. L. Lingle, S. L. Barrett, V. C. Negron, A. B. D'Assoro, K. Boeneman, W. Liu, C. M. Whitehead, C. Reynolds, and J. L. Salisbury
Centrosome amplification drives chromosomal instability in breast tumor development
PNAS, February 19, 2002; 99(4): 1978 - 1983.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (89K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (175)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Liu, W.
Right arrow Articles by James, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, W.
Right arrow Articles by James, C. D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?