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Pyrosequencing analysis of KRAS codon 61 mutations in Thai patients with advanced colorectal cancer

Chinachote Teerapakpinyo
Chinachote Teerapakpinyo
, 
Phanni Wanthong
Phanni Wanthong
, 
Mathawee Aumchaaumchaya
Mathawee Aumchaaumchaya
, 
Piyamai Chankate
Piyamai Chankate
, 
Warisa Kaikeaw
Warisa Kaikeaw
, 
Warunya Tosakorn
Warunya Tosakorn
 e 
Shanop Shaungshoti
Shanop Shaungshoti
   | 31 gen 2017
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Article Category: Brief communication (Original)
Pubblicato online: 31 gen 2017
Pagine: 61 - 67
DOI: https://doi.org/10.5372/1905-7415.0901.369
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© 2017 Chinachote Teerapakpinyo, Phanni Wanthong, Mathawee Aumchaaumchaya, Piyamai Chankate, Warisa Kaikeaw, Warunya Tosakorn, Shanop Shaungshoti
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

PCR amplification of exon 3 (codon 61) in KRAS. Codon 61 (CAA, nucleotides 181–183), which resides in exon 3 of KRAS (Transcript ID: ENST00000311936; Ensembl database) is shown in the square box. Mutations in nucleotides 182 and 183 (yellow highlighted nucleotide) reportedly result in amino acid changes including c.182A>T (p.Q61L), c.182A>G (p.Q61R), c.182A>C (p.Q61P), c.183A>C (p.Q61H), and c.183A>T (p.Q61H) in the Catalogue Of Somatic Mutations In Cancer (COSMIC) database, version 63 release. Nucleotides 182 and 183 in codon 61 were sequenced in the reverse direction using KRAS61-PySeq1 and KRAS61-PySeq2 primers. A: KRAS61-Pyseq1 (5′-CTGGTCCCTCATTGCACTGTACTC-3′) and B: KRAS61-Pyseq2 (5′-GTCCCTCATTGCACTGTACTCCTCT-3′) primers complementary to red underlined nucleotides were used for pyrosequencing.
PCR amplification of exon 3 (codon 61) in KRAS. Codon 61 (CAA, nucleotides 181–183), which resides in exon 3 of KRAS (Transcript ID: ENST00000311936; Ensembl database) is shown in the square box. Mutations in nucleotides 182 and 183 (yellow highlighted nucleotide) reportedly result in amino acid changes including c.182A>T (p.Q61L), c.182A>G (p.Q61R), c.182A>C (p.Q61P), c.183A>C (p.Q61H), and c.183A>T (p.Q61H) in the Catalogue Of Somatic Mutations In Cancer (COSMIC) database, version 63 release. Nucleotides 182 and 183 in codon 61 were sequenced in the reverse direction using KRAS61-PySeq1 and KRAS61-PySeq2 primers. A: KRAS61-Pyseq1 (5′-CTGGTCCCTCATTGCACTGTACTC-3′) and B: KRAS61-Pyseq2 (5′-GTCCCTCATTGCACTGTACTCCTCT-3′) primers complementary to red underlined nucleotides were used for pyrosequencing.

Figure 2

Histograms for KRAS codon61 wild-type and mutant discriminations. Histograms (A–G) were generated by PyroMark Q96 software using dispensation order and sequence to analyze as follows; (A–C) 5′-GCTCGATC GA-3′ and 5′-CTC[T/G/A]TGACCTGATGT-3′, (D–G) 5′-TACGTACTGC-3′ and 5′-[T/A/C/G]GACCTGCTG TG-3′, respectively.
Histograms for KRAS codon61 wild-type and mutant discriminations. Histograms (A–G) were generated by PyroMark Q96 software using dispensation order and sequence to analyze as follows; (A–C) 5′-GCTCGATC GA-3′ and 5′-CTC[T/G/A]TGACCTGATGT-3′, (D–G) 5′-TACGTACTGC-3′ and 5′-[T/A/C/G]GACCTGCTG TG-3′, respectively.

Figure 3

Pyrograms showing mutations in KRAS codon 61. (A) Heterozygous c.182A>T (p.Q61L); (B) heterozygous c.183A>T (p.Q61H) and (C) heterozygous c.183A>C (p.Q61H) in samples from Thai patients with advanced colorectal cancer. The red arrows indicate mutant peak and the percentage of mutant allele in the amplified PCR products. wt: wild type; mut: mutant
Pyrograms showing mutations in KRAS codon 61. (A) Heterozygous c.182A>T (p.Q61L); (B) heterozygous c.183A>T (p.Q61H) and (C) heterozygous c.183A>C (p.Q61H) in samples from Thai patients with advanced colorectal cancer. The red arrows indicate mutant peak and the percentage of mutant allele in the amplified PCR products. wt: wild type; mut: mutant
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1875-855X
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Inglese
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Medicine, Assistive Professions, Nursing, Basic Medical Science, other, Clinical Medicine
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