Loss of heterozygosity on chromosome 10q22-10q23 and 22q11.2-22q12.1 and p53 gene in primary hepatocellular carcinoma

Table 1 PCR amplification primer pairs for p53 gene, 10q22-10q23 and 22q11.2-22q12.1

Markers name	Forward	Reverse	Annealing (T °C)	Size (bp)
TP53.A1/TP53.A2 (p53 gene exon 2-3)	TGGATCCTCTTGCAGCAGCC	AACCCTTGTCCTTACCAGAA	54	270
TP53.B1/TP53.B2 (p53 gene exon 4)	ATCTACAGTCCCCCTTGCCGGC	AACTGACCGTGCAAGTCA	57	296
TP53.G1/TP53.G2 (p53 gene exon 11)	TCTCCTACAGCCACCTGAAG	CTGACGCACACCTATTGCAA	58	122
D10S579	CCGATCAATGAGGAGTGCC	ATACACCCAGCCAATGCTGC	60	260
D22S421	CTGCTGCCCCTAACATATCAC	GGCCAGGAGTGTCTGAATTTTA	65	163
CDK41	GGAGGTCGGTACCAGAGTG	CATGTAGACCAGGACAGG	60	364

¹The aim of PCR amplification of CDK4 gene was to confirm that genomic DNA had been truly extracted from all samples. These primer sequences were retrieved from the Genome Database (http://gdbwww.gdb.org). PCR amplification was performed in a 50 μL reaction volume containing 400 ng template DNA, 0.2 mmol/L of each deoxynucleotide triphosphate, 20 mmol/L of each primer, 1.5 mmol/L MgCl₂, 1 × reaction buffer and 2 U Taq DNA polymerase. The reaction mixture was denatured for 5 min at 94 °C. DNA was subsequently amplified for 35 cycles with 94 °C for 30 s, 54-65 °C for 30 s, 72 °C for 40 s, and a final extension at 72 °C for 8 min. PCR product (8 μL) was electrophoresed in a 20 g/L agarose gel, visualized by staining with ethidium bromide and ultraviolet illumination, and documented by a computer-linked camera. The target DNA fragments were confirmed by compar-ing to a 100 bp DNA ladder.

Table 2 Clinical and genetic features of 20 patients with HCC (*, LOH; △, homozygous deletion; O, no deletion; +, HBsAg positive; -, HBsAg negative)

Case No.	Age (yr)	Sex	HBsAg	TP53.A	TP53.B	TP53.G	D10S579	D22S421
1	49	M	+	*	*	O	*	O
2	55	M	+	O	△	O	O	△
3	39	M	+	O	△	O	△	△
4	55	F	+	*	O	O	O	*
5	72	F	+	O	*	△	△	O
6	40	M	+	O	O	O	O	O
7	34	F	+	O	*	O	△	△
8	56	M	+	O	O	O	O	O
9	27	F	-	O	*	O	△	O
10	50	M	+	O	O	O	O	△
11	48	M	+	O	△	O	△	△
12	52	M	+	O	O	O	△	O
13	63	F	+	O	△	△	△	△
14	60	M	+	O	△	O	*	△
15	65	M	+	O	O	O	*	O
16	34	M	+	O	*	O	O	O
17	64	M	+	*	*	O	*	*
18	52	F	+	O	O	O	△	O
19	32	F	+	O	△	O	O	△
20	38	M	+	*	O	O	O	*
LOH rate (%)				20	30	0	20	15
Homozygous deletion rate (%)				0	30	10	40	40