Printed in the Republic of Korea
DHPLC p53
*
(2004. 1. 14 )
Analysis of p53 Somatic Mutation in Head and Neck Cancer Using Denaturing High Performance Liquid Chromatography(DHPLC)
Kwang-Youl Kim, Sang-Bum Park, Sang-Man Han, Youn-Hyoung Nam, and Won-Cheoul Jang* Department of Chemistry, College of Natural Science, Dankook University, Cheonan 330-714, Korea
(Received January 14, 2004)
. (HNSCC: head and neck squamous cell carcinoma) p53
(tumor suppressor gene) !"#$ %& '() *+*$ ,-) ./ 0/ 12. 34 56 -) 7 8/ 9 8& : ;< 50=> ?-) p53 exon 5-8 @A
BC56 DNA> DE PCR-SSCP(polymerase chain reaction single strand conformational polymorphism) F G DHPLC(denaturing high performance liquid chromatography) FG-) p53H !"#(somatic mutation)
> ' IJK2. L M SSCP IJ FG& 16=(32%), DHPLC IJ FG& 17=(34%) > NEK/ L O SSCPP DHPLC IJ FG Q exon 8R56 MS(deletion) TU !"#> VWK-X YZ-) [ \ ] 6^ IJ](automatic DNA sequencer) > _ Q` !"#> VWK2. DHPLC IJFG# SSCP FG .2 IJ ab#* cd# e fQ0X .2 g hVi !"# NE FGjk VWK2.
: HNSCC, p53, DHPLC, PCR-SSCP
ABSTRACT. Mutation of p53 tumor suppressor gene in HNSCC (head and neck squamous cell carcinoma) has been proposed high rate. We extracted genomic DNA from 50 head and neck cancer. The DNA was amplified by PCR at exon 5-8 in p53 tumor suppressor gene. We have compared single strand conformation polymorphism (SSCP) and denaturing high performance liquid chromatography (DHPLC) method for analysis of p53 somatic mutation. As a result, 16 deleted mutations (32%) were detected by SSCP analysis and 17 deleted mutations (34%) were detected by DHPLC analysis at exon 8. All of 17 mutations were proved by sequencing. We conclude that DHPLC is a fast and simple screening method rather than SSCP analysis.
Keywords: HNSCC, p53, DHPLC, PCR-SSCP
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Table 1. Primer sequence used to amplify p53 tumor suppressor gene.
Sequence of primer Length(bp) Position
exon5 5'-ctcttcctacagtactcccctgc-3'
211 1543-1753
5'-gccccagctgctcaccatcgcta-3' exon6 5'-gattgctcttaggtctggcccctc-3'
182 1808-1989
5'-ggccactgacaaccacccttaacc-3' exon7 5'-gtgttatctcctaggttggctctg-3'
139 2487-2625
5'-caagtggctcctgacctggagtc-3' exon8 5'-acctgatttccttactgcctctggc-3'
200 2906-3105
5'-gtcctgcttgcttacctcgcttagt-3'
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Fig. 1. SSCP analysis of fragments of the p53 gene. Three of the 10 band shifts reconized by PCR-SSCP are shown here. DNA samples of head and neck tumors and normal tis- sue were amplified using PCR primers for the exon 8.
N; normal control tissue, Lanes 1-3, 5-6, 8 ; wild-type, lanes 4, 7, 9 ; mutant-type.
Fig. 2. Impact of column temperature on the resolution homoduplex(A), and heteroduplex(B) in HNSCC.
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Fig. 3. Chromatograms produced DHPLC analysis of wild type and mutant type in HNSCC.
Fig. 4. Automatic sequencing analysis of exon 8 in HNSCC.
A) wild type, B) mutant type (14484del T)
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