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Technical information

SNPs typing - SNP typing on human genome

Procedures

SNP typing on cloned DNA

SNP typing on human genome

SNP typing using fluorescent intercalating dye

			The CYP2C19 gene and its family genes

In the case of SNP typing for CYP2C19 that relates to drug metabolism, only the target sequence of CYP2C19 has to be amplified among similar sequences of highly homologous genes (left figure), and furthermore, the type of SNP has to be distinguished. Since SNP typing requires very high specificity, it is extremely difficult for the reaction methods using only two types of primer and probe. However, as illustrated in this example, SNP typing can be specifically done by LAMP method as it uses 4 primers to recognize 6 distinct regions.

			SNP typing on CYP2C19 using human genome specimens

SNP typing on CYP2C19 using human genome specimens

The above data was obtained from the SNPs typing of human genome specimens on CYP2C19^*2 allele (left) and CYP2C19^*3 allele (right). This figure represents the fluorescence intensity from the reaction of Wild Type (WT) primers (horizontal axis) and the reaction of Mutant Type (MUT) primers (vertical axis) , respectively.
As a result, the following 3 types have been clearly distinguished by the respective primers: WT homogeneous samples can only be amplified by WT primers. MUT homogeneous samples can only be amplified by MUT primers. And hetero samples can be amplified by both WT and MUT primers.

*Data was collected from voluntary tests among company employees.

			Comparison with the PCR-RFLP method

	PCR-RFLP
LAMP	^1/^1	^1/^2	^1/^3	^2/^2	^2/^3	^3/^3
^1/^1	30	0	0	0	0	0
^1/^2	0	25	0	0	0	0
^1/^3	0	0	14	0	0	0
^2/^2	0	0	0	9	0	0
^2/^3	0	0	0	0	6	0
^3/^3	0	0	0	0	0	2

n=86

The table shows the result of SNP typing of the CYP2C19 genotype. The results agreed with those from PCR-RFLP using the same samples. The data was also confirmed by sequencing result.
However, while the conventional PCR-RFLP method involves three different steps and takes about 5 hours, LAMP-based SNP typing on the CYP2C19 gene can be achieved in a single step within 30 minutes.

Validation of the Loop-Mediated Isothermal Amplification Method for Single Nucleotide Polymorphism Genotyping with Whole Blood
Masaomi Iwasaki, Toshihiro Yonekawa, et al. Genome Letters, Vol.2 (3) 119-126, 2003

		LAMP standard procedures

		Detection methods

		SNPs typing

Quantification method