DNA fragment preparation method for gene expression profiling

A DNA fragment preparation method for gene expression profiling comprising the steps of:

• i) preparing a plurality of double stranded DNA fragments including a sequence-known DNA fragment and a sequence-unknown DNA fragment from a sample DNA;
• ii) ligating an oligomer having a known base sequence to one 3′-terminus of the double stranded DNA fragments by a ligation reaction to obtain a ligated sequence-known DNA fragment and a ligated sequence-known DNA fragment;
• and
• iii) by a polymerase chain reaction (PCR), amplifying double stranded fragments each having a shorter length than the length of the ligated sequence-known DNA fragment and the ligated sequence-unknown DNA fragment, using the ligated sequence-known DNA fragment and the ligated sequence-unknown DNA fragment as templates and using a first primer which hybridizes with a base sequence at the 3′-terminus of one strand, being not ligated to the oligomer, of the ligated sequence-known DNA fragment and the ligated sequence-unknown DNA fragment, a second primer which hybridizes with the known base sequence of the oligomer of the ligated sequence-known DNA fragment and the ligated sequence-unknown DNA fragment, and a specific primer that hybridizes specifically with a base sequence complementary with one strand, being ligated to the oligomer, of the ligated sequence-known DNA fragment, at a position between a base sequence complementary with the first primer and a base sequence complementary with the second primer,
• wherein, by using the first primer, the second primer, and the specific primer at the same time, and by competitive reactions occurring between PCR by the first primer and the second primer and PCR by the second primer and the specific primer, the double stranded fragments each having a part of the base sequence of the sequence-known DNA fragment and having a shorter length than lengths of PCR products by the first primer and the second primer are produced as major products in the PCR by the second primer and the specific primer.

A DNA fragment preparation method for gene expression profiling comprising the steps of:

• i) preparing a plurality of double stranded cDNA fragments including a sequence-known cDNA fragment and a sequence-unknown cDNA fragment from sample mRNAs, the double stranded cDNA fragments each consisting of a first single strand having a poly A sequence at the 3′ terminus and a second single strand having a poly T sequence at the 5′ terminus;
• ii) ligating an oligomer having a known base sequence to the 3′-terminus of the second single strand of the double stranded cDNA fragments by a ligation reaction to obtain a ligated sequence-known cDNA fragment and a ligated sequence-unknown cDNA fragment;
• and
• iii) by polymerase chain reaction (PCR), amplifying double stranded fragments each having a shorter length than the length of the ligated sequence-known cDNA fragment and the ligated sequence-unknown cDNA fragment, using the ligated sequence-known cDNA fragment and the ligated sequence-unknown cDNA as templates and using a first primer which hybridizes with the poly A sequence at the 3′ terminus of the first single strand of the ligated sequence-known cDNA fragment and the ligated sequence-unknown cDNA, a second primer which hybridizes with the known base sequence of the oligomer of the second single strand of the ligated sequence-known cDNA fragment and the ligated sequence-unknown cDNA, and a specific primer that hybridizes specifically with a base sequence of the first single strand of the ligated sequence-known cDNA fragment at a position between a base sequence complementary with the first primer and a base sequence complementary with the second primer,
• wherein, by using the first primer, the second primer, and the specific primer at the same time, and by competitive reactions occurring between PCR by the first primer and the second primer and PCR by the second primer and the specific primer, the double stranded fragments each having a part of the base sequence of the sequence-known DNA fragment and having a shorter length than the lengths of PCR products by the first and second primers are produced as major products in the PCR by the second primer and the specific primer.