Table 2.
Primer and probe nucleotide sequences used for the detection of Y chromosome and HA-1H–positive microchimeric cells by real-time PCR
| Primer | Nucleotide sequence | Fragment size, bp |
|---|---|---|
| Forw-DYS1 | 5′-TCCTGCTTATCCAAATTCACCAT-3′ | 86* |
| Rev-DYS1 | 5′-ACTTCCCTCTGACATTACCTGATAATTG-3′ | |
| Y probe | 5′-(FAM)-AAGTCGCCACTGGATATCAGTTCCCTTGT-(TAMRA)-3′ | |
| Forw-HCK | 5′-TATTAGCACCATCCATAGGAGGCTT-3′ | 81* |
| Rev-HCK | 5′-GTTAGGGAAAGTGGAGCGGAAG-3′ | |
| HCK Probe | 5′-(FAM)-TAACGCGTCCACCAAGGATGCGAA-(TAMRA)-3′ | |
| Forw-I-COM | 5′-GACGTCGTCGAGGACATCTCCCATC-3′ | 324† |
| Rev-I HA-1H | 5′-CATCAGATCCTTTAAAAAAAGTGG-3′ | |
| For-II HA-1H | 5′-CTTAAGGAGTGTGTGCTGCA-3′ | 191‡ |
| HA-1H Rev-II | 5′-ACTCCTACACATCCCTCAGA-3′ | |
| For-I HCK | 5′-ACCTCCCCGAAGATTCAGAC-3′ | 381† |
| Rev-I HCK | 5′-TTGGGGGCAAGTTGAGTTTA-3′ | |
| For-II HCK | 5′-TATTAGCACCATCCATAGGAGGCTT-3′ | 81‡ |
| Rev-II HCK | 5′-GTTAGGGAAAGTGGAGCGGAAG-3′ |
*
Amplification conditions: 95°C for 10 minutes, then 40 cycles at 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds.
†
Amplification reactions: 5 minutes at 95°C, then 30 cycles at 95°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute. The cycling was followed by 72°C for 5 minutes.
‡
Amplification conditions: 4.5 minutes at 95°C, then 5 cycles at 95°C for 15 seconds and 66°C for 45 seconds, followed by 35 cycles at 95°C for 15 seconds, 62°C for 45 seconds, and 72°C for 30 seconds.