US 11,718,882 B2
EML4-ALK gene mutation analysis method
Byung Hee Jeon, Seongnam-si (KR)
Assigned to CYTOGEN, INC., Seoul (KR)
Appl. No. 16/631,324
Filed by CYTOGEN, INC., Seoul (KR)
PCT Filed Jul. 17, 2018, PCT No. PCT/KR2018/008046
§ 371(c)(1), (2) Date Jan. 15, 2020,
PCT Pub. No. WO2019/031722, PCT Pub. Date Feb. 14, 2019.
Claims priority of application No. 10-2017-0099591 (KR), filed on Aug. 7, 2017.
Prior Publication US 2020/0140959 A1, May 7, 2020
Int. Cl. C12Q 1/6886 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6827 (2018.01)
CPC C12Q 1/6886 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6827 (2013.01); C12Q 2549/119 (2013.01); C12Q 2600/106 (2013.01); C12Q 2600/158 (2013.01); G01N 2800/7028 (2013.01)] 13 Claims
 
1. 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.