Patent RU-2631797-C2 (RU)
Inventor: ROBINS KHARLAN S
IPC: C12Q1/68
Filed: 2013-05-08
Granted: 2017-09-26
Abstract: FIELD: biotechnology. 
     SUBSTANCE: composition for standardizing the amplification efficiency of a set of oligonucleotide primers for amplifying rearranged nucleic acid sequences encoding one or more receptors of the adaptive immune system in a biological sample received from lymphoid cells of a mammalian subject is described, each receptor of the adaptive immune system comprises a variable region and a connecting site. The composition comprises: a plurality of synthetic matrix oligonucleotides. Each synthetic matrix oligonucleotide has a known concentration before amplification and oligonucleotide sequence of the common formula: 5'-U1-B1-V-B2-R-B3-J-B4-U2-3' [I], and: (a) V represents an oligonucleotide sequence containing at least 20 and not more than 1000 consecutive nucleotides of gene sequence encoding a variable (V) spot of the receptor of the adaptive immune system or its complement, and each V contains a unique oligonucleotide sequence of the V-spot; (b) J is an oligonucleotide sequence containing at least 15 and not more than 600 consecutive nucleotides of gene sequence, which encodes the connective (J) spot of the receptor of the adaptive immune system or its complement, and each J contains a unique oligonucleotide sequence of J-spot; (C) U1 is either absent or contains an oligonucleotide sequence selected from (i) the first universal sequence of the oligonucleotide adapter, and (ii) the first specific for sequencing platform of the oligonucleotide sequence which is linked to position 5' and is located in it relative to the universal sequence of the oligonucleotide adapter; (d) U2 is either absent or contains an oligonucleotide sequence selected from (i) the second universal sequence of the oligonucleotide adapter and (ii) the second specific for sequencing platform oligonucleotide sequence which is linked to position 5' and is located in it relative to the second universal sequence of the oligonucleotide adapter; (e) at least one of B1, B2, B3, and B4 are present and each of B1, B2, B3 and B4 contains the oligonucleotide B containing a sequence of barcode from 3-25 consecutive nucleotides which identifys uniquely as a paired combination, (i) the unique oligonucleotide sequence of the V-spot from (a) and (ii) the unique oligonucleotide sequence of the J-spot from (b); (f) R is either absent or contains a recognition site of the restriction enzyme, which contains the oligonucleotide sequence which is absent in (a)-(e); and moreover: (g) the plurality of synthetic matrix oligonucleotides contains a quantity of at least a or at least b of the unique oligonucleotide sequences, depending on whichever is greater, and a represents the number of unique segments of the gene encoding the V-spot of the receptor of the adaptive immune system of the subject, and b represents the number of unique segments of the gene encoding the J-spot of the receptor of the adaptive immune system of the subject, and the composition contains at least one synthetic matrix oligonucleotide for each unique oligonucleotide sequence of the V-spot and at least one synthetic matrix oligonucleotide for each unique oligonucleotide sequence of the J-spot. A method for quantifying a plurality of rearranged nucleic acid molecules encoding one or a plurality of receptors of an adaptive immune system in a biological sample is also claimed. 
     EFFECT: increase the accuracy of measurements. 
     27 cl, 14 dwg, 17 tbl, 12 ex