EML4-ALK gene mutation analysis method
AssigneeCytogen Corporation
InventorByung Hee Jeon
Life of this patent
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Abstract
qRT-PCR primers capable of detecting EML4-ALK gene variant based on circulating tumor cells at a more sensitive detection limit than a conventional method. Also disclosed is a lung cancer patient screening method which is capable of detecting EML4-ALK gene variant using circulating tumor cells even in a lung cancer patient on whom ALK-FISH testing has been difficult to perform, due to difficulty in collecting a solid lung cancer tissue sample, and is able to determine whether an anticancer drug targeting the EML4-ALK gene variant may be applied to the lung cancer patient.
Claims13
A method for analyzing EML4-ALK gene variant comprising the steps of:
- (a) obtaining a liquid biopsy sample from a cancer patient;
- (b) isolating circulating tumor cells from the liquid biopsy sample using a biochip;
- (c) performing short-term culture of the isolated circulating tumor cells in the step (b);
- (d) isolating RNA from the short-term cultured circulating tumor cells in the step (c);
- (e) performing qRT-PCR using the isolated RNA as a template and two qRT-PCR primers;
- (f) performing nested PCR using a resulting product from the qRT-PCR as a template and two nested primers for the two qRT-PCR primers;
- and
- (g) detecting EML4-ALK gene variant type based on the resulting product from the nested PCR,
- wherein the biochip is a high-density microporous chip coated with a BSA solution,
- wherein the high-density microporous chip is a size-based chip,
- wherein a pore size of the high-density microporous chip is 5.5 to 8.5 μm,
- wherein the circulating tumor cells in the step (b) is isolated by gravity,
- wherein the culture medium used in the short-term culture is consisting of insulin, transferrin, epidermal growth factor (EGF) and an Rho kinase (ROCK) inhibitor, and
- wherein the culture plate used in the short-term culture is coated with hydrogel.
Dependencies are inferred from the claim text.