Wako Pure Chemical Industries, Ltd.Wako Pure Chemical Industries, Ltd. - Laboratory Chemicals
Home > Products > Life Science > Genetic Research > LH Gene Mutation Detection Kit series

Detects gene mutations simply, rapidly, accurately, inexpensively!!
Gene Mutation Detection Kit series

LH Gene Mutation Detection Kit series

遺伝子変異検出キット Point mutations, insertion mutations and deficient mutations in target genes (BRAF, EGFR, KRAS) can be detected simply, rapidly, accurately, and inexpensively.
Regarding Loop Hybrid Mobility Shift Assay (LH-MSA method) as a basic principle, the secondary structural change of Loop Hybrid Probe (LH Probe) hybridizing in mutation gene regions is detected in polyacrylamide gel electrophoresis.
LH-MSA method does not require special facilities and equipments, such as expensive real-time PCR equipment and specific probes.


1. Link


2. Features

  • Extremely inexpensive compared to real-time PCR method
  • Special facilities and equipments are not required
  • Operation is simple, work time and labor are reduced
  • Rare mutation is detectable by 1 probe.
  • Very high sensitivity

3. Operation Outline / Detection Principle

Operation Outline

Detection Principle

4. Product List

Product Name Package Size Wako Catalog No. Storage Condition
LH Gene Mutation Detection Kit
for BRAF V600E   Package Insert
1 kit
(16 samples x for 6 tests)
299-76101 Keep at -20°C.
LH Gene Mutation Detection Kit
for EGFR Exon19 Deletion   Package Insert
1 kit
(16 samples x for 6 tests)
291-76301
LH Gene Mutation Detection Kit
for EGFR Exon20 Insertion   Package Insert
1 kit
(16 samples x for 6 tests)
293-76501
LH Gene Mutation Detection Kit
for EGFR Exon21 L858R   Package Insert
1 kit
(16 samples x for 6 tests)
295-76701
LH Gene Mutation Detection Kit
for KRAS Mutations at Codons 12 and 13   Package Insert
1 kit
(16 samples x for 6 tests)
297-76901

5. Applications - Electrophoresis

Using genomic DNA extracted from frozen sections of human colon cancer tissues or formamide, paraffin-embedded sections of human lung cancer tissues and other control DNA, gene mutations were detected in each of target genes (BRAF, EGFR, KRAS).
This application data is provided by Shoichi Matsukuma, Kanagawa Cancer Center Research Institute, division of molecular pathology and genetics.


Application 1 - Detection of point mutation of BRAF genes

Using samples shown in experimental conditions and the other control DNA, point mutation was detected in BRAF gene codone 600.

【Results】

  • A band of mutant samples (sample 4~6) was detected with a high sensitivity in the same position as BRAF V600E mutant cell line (COLO 205) !!
  • Also detects rare mutation of D594G (Sample 7) also by the same LH Probe!!
WakoReport Anticancer agent Zelboraf has been turned out to be highly effective in patients with mutations in BRAF genes in clinical trials. It was approved as the treatment drug in the case of that there are BRAF V600E gene mutations of unresectable or metastatic melanomas by the US Food and Drug Administration (FDA) in August 2011.
Since there are BRAF genes in about 10 % of colon cancers and genomic DNA of colon cancer patients who have mutations in BRAF genes is a high methylation state, numerous research studies on the accumulation of methylation and gene mutations have been reported.

[Experimental conditions]
Sample 1-7: Genomic DNA derived from frozen sections of human colon cancer tissues
· Electrophoresis gel: SuperSep ™ Ace, 10%, 17well (recommended product)
[Constant current, 20mA]
· Electrophoretic time: 70 minutes (standard)
· Staining: Gel Red ™ Nucleic Acid Gel Stain, 10,000x in water
BRAF gene PCR amplified product: 157 bp

Application 2 - Detection of Exon19 deficient mutation of EGFR genes

Using samples shown in experimental conditions and other control DNA, deficient mutation was detected in codon 746-750 of EGFR gene Exon19.

【Results】

  • Detects the band shift of mutant sample with a high sensitivity in even genomic DNA extracted from FFPE sections * 1 of the worst quality!!
  • Detects rare mutation of insertion mutation(sample 8) by the same LH Probe!!

* 1 FFPE: formalin-fixed, paraffin-embedded

WakoReport Gefitinib has the action of inhibiting the function of tyrosine kinase which is an Epidermal Growth Factor Receptor (EGFR) and was approved as a molecule-targeting therapeutic agent to non-small cell lung cancer (NSCLC) in Japan first in the world in July 2002.
However, the occurrence of interstitial pneumonitis due to this drug has become a major problem as a side effect. Gefitinib was confirmed to be effective when there are genetic mutations in the tyrosine kinase active sites of EGFR, and the inspection has become important.
It has been reported that there are deficient mutations in the site centered Exon19 codon 746-750 of EGFR gene of NSCLC of 20 ~ 25%.

[Experimental conditions]
Sample 1-8: Genomic DNA derived from formamide, paraffin-embedded sections of human lung cancer tissues
· Electrophoresis gel: SuperSep ™ Ace, 10%, 17well (recommended product)
[Constant current, 20mA]
· Electrophoretic time: 70 minutes (standard)
· Staining: Gel Red ™ Nucleic Acid Gel Stain, 10,000x in water
EGFR gene Exon19 PCR amplified product: 146 bp"

Application 3 - Detection of Exon20 insertion mutation of EGFR genes

Using samples shown in experimental conditions and other control DNA, insertion mutation has been detected in EGFR gene Exon20.

【Result】

  • Detects the band shift of mutant sample with a high sensitivity in even genomic DNA extracted from FFPE sections of the worst quality!!
WakoReport Gefitinib has the action of inhibiting the function of tyrosine kinase which is an Epidermal Growth Factor Receptor (EGFR) and was approved as a molecule-targeting therapeutic agent to non-small cell lung cancer (NSCLC) in Japan first in the world in July 2002.
However, the occurrence of interstitial pneumonitis due to this drug has become a major problem as a side effect. Gefitinib was confirmed to be effective when there are genetic mutations in the tyrosine kinase active sites of EGFR, and the inspection has become important.
It has been reported that there are insertion mutations in EGFR gene Exon20 of NSCLC of about 5%.

[Experimental conditions]
Sample 1-7: Genomic DNA derived from formamide, paraffin-embedded sections of human lung cancer tissues
· Electrophoresis gel: SuperSep ™ Ace, 10%, 17well (recommended product)
[Constant current, 20mA]
· Electrophoretic time: 70 minutes (standard)
· Staining: Gel Red ™ Nucleic Acid Gel Stain, 10,000x in water
EGFR gene Exon20 PCR amplified product: 189 bp

Application 4 - Detection of Exon21 point mutation of EGFR genes

Using samples shown in experimental conditions and other control DNA, L858R point mutation of EGFR gene Exon21 was detected.

【Result】

  • Detects the band shift of mutant sample with a high sensitivity in even genomic DNA extracted from FFPE sections of the worst quality!!
WakoReport Gefitinib has the action of inhibiting the function of tyrosine kinase which is an Epidermal Growth Factor Receptor (EGFR) and was approved as a molecule-targeting therapeutic agent to non-small cell lung cancer (NSCLC) in Japan first in the world in July 2002.
However, the occurrence of interstitial pneumonitis due to this drug has become a major problem as a side effect.
Gefitinib was confirmed to be effective when there are genetic mutations in the tyrosine kinase active sites of EGFR, and the inspection has become important.
It has been reported that there are point mutations from leucine to arginine in Exon21 codon 858 of EGFR gene of NSCLC of 20~25%.

[Experimental conditions]
Sample 1-4: Genomic DNA derived from formamide, paraffin-embedded sections of human lung cancer tissues
· Electrophoresis gel: SuperSep Ace, 10%, 17well (recommended product)
[Constant current, 20mA]
· Electrophoretic time: 70 minutes (standard)
· Staining: Gel Red™ Nucleic Acid Gel Stain, 10,000x in water
EGFR gene Exon21 PCR amplified product: 184 bp

Application 5 - Detection of point mutation of KRAS genes

Using samples shown in experimental conditions and other control DNA, a point mutation was detected in glycine of codon 12 and 13 of KRAS genes.

【Results】

  • Detects the band shift of the mutant sample (sample 2-7) with a high sensitivity !!
  • Also detects mutations other than G12D by the same LH Probe!!
WakoReport Anti-cancer drug cetuximab has been found to be highly effective to the wild-type patients without mutations in KRAS genes in clinical trials, and the Ministry of Health, Labour and Welfare instructed to append that it should be administered taking the presence or absence of KRAS gene mutation into account in the notification issued on March 23, 2010.
Since there are KRAS gene mutations in about 30% of colon cancer patients, and genomic DNA of colon cancer patients who have mutations in KRAS genes is medium-methylated state, numerous research studies on the accumulation of methylation and gene mutations have been reported.
[Experimental conditions]
Sample 1-7: Genomic DNA derived from frozen sections of human colon cancer tissues
· Electrophoresis gel: SuperSep Ace, 10%, 17well (recommended product)
[Constant current, 20 mA]
· Electrophoretic time: 70 minutes (standard)
· Staining: Gel Red™ Nucleic Acid Gel Stain, 10,000x in water
KRAS gene PCR amplified product: 165 bp

6. Kit Components (for 96 samples: 16 samples x 6 tests)

1. 10 x Buffer ········ 102 μL x 1
2. dNTPs ·············· 102 μL x 1
3. DNA Polymerase ··· 12 μL x 1
4. Forward Primer*1··· 51 μL x 1
5. Reverse Primer*1······· 180 μL x 1
6. LH Probe·················· 96 μL x 1
7. Wild Type Control DNA··· 6 μL x 1
8. Each Control DNA*2······· 6 μL x 1
  9. Loading Buffer*3··· 288 μL x 1
10. DNA Marker············ 9 μL x 1
*1: for Exon21 L858R: Forward Primer 180 μL, Reverse Primer 51 μL
*2: BRAF V600E: V600E control DNA, EGFR Exon19: deficient-type control DNA, EGFR Exon20: insertion-type control DNA
     EGFR Exon21: L858R control DNA, KRAS Mutations: G12D control DNA
*3: The content is 6x concentration. In addition, the blue and orange dyes are included.

7. Related Products

Product Name Package Size Wako Catalog No. Storage Condition
SuperSep Ace, 10%, 17well 10 EA 192-14961 Keep at 2-10°C.
SuperSep Ace, 12.5%, 17well 10 EA 196-14981
10 x Tris-Glycine Buffer 1 L 201-18601
Distilled Water, Deionized, Sterile
Manufactured by Nippon Gene
100 mL 316-90101 Keep at RT.
EasySeparator
(an Electrophoresis tank for SuperSep precast gel)
1 set 058-07681 -
DNA Isolator PS Kit 100 tests 295-52401 Keep at 2-10°C.
Home > Products > Life Science > Genetic Research > LH Gene Mutation Detection Kit series