Table 2.
KIR locus specific polymerase chain reaction amplification primersa and conditions
| KIR Locus |
Amplicon | Sense Primer | Antisense Primer | Annealing Sites-- Sense/Antisenseb |
Amplicon Size (bp) |
PCR Reaction Conditions | PCR Reaction Components (50 µl) | |||
|---|---|---|---|---|---|---|---|---|---|---|
| Annealing Temp (° C)—Initial/ Secondary Cycles |
Extension Time (min) |
MgSO4 (µl) |
DMSO/ Betaine (µl) |
Taq | ||||||
| KIR2DL1 | A | TGTAAAACGACGGCCAGTGGCAGCACCATGTCGCTCT | CAAGCAGTGGGTCACTTGAC | 10T/633G | 5605 | 64/61 | 5 | 2.0 | 2.5/− | High Fidelity |
| A2 | ATAACATCCTGTGCGCTGCT | GGGTCACTGGGAGCTGACAC | 5UTR/331G | 3825 | 66/64 | 5 | 1.5 | 3.0/− | High Fidelity | |
| B | ACTCACTCCCCCTATCAGG | TGTTGACTCCCTAGAAGACG | 331G/3UTR | 10282 | 62/59 | 10 | 2.0 | −/− | High Fidelity | |
| KIR2DL2 | A | TCTCAGCACAGACAGCACC | GCCCTGCAGAGAACCTACA | 5UTR/505T | 5382 | 62/58 | 7 | 2.0 | 2.0/− | High Fidelity |
| B | CCATGATGGGGTCTCCAAA | TCAATGCCTGCATCGAAGGTTTCT | 246A/IN6 | 5348 | 60/57 | 5 | 3.5 | −/10 | High Fidelity | |
| C | TCACCCACTGAACCAAGCTCT | TGTTGACTCCCTAGAAGACG | 708T/3UTR | 5228 | 62/58 | 7 | 2.0 | 2.0/− | High Fidelity | |
| D | AATGCCTCTTCTCCTCCAGGTCTA | CTCTCCTCTGGGTCTCTCCTGACCG | 375A/IN5 Nested Ex5 | 568 | 62/57 | 1.5 | - | −/10 | Taq/10X PCR buffer with MgCl2 | |
| KIR2DL3 | A | TGTAAAACGACGGCCAGTGGCAGCACCATGTCGCTCA | GCCCTGCAGAGAACCTACG | 10A/505C | 5385 | 62/58 | 5 | 3.0 | −/10 | High Fidelity |
| B1 | GTTCTGTTACTCACTCCCCCT | CTCTCCTCTGGGTCTCTCCTGACCG | 325T/IN5 | 2131 | 62/58 | 5 | 3.0 | −/10 | High Fidelity | |
| B2 | CGTTCTGCACAGAGAAGGGAAc | 194A/IN5 | 2262 | 62/58 | 5 | 3.0 | −/10 | High Fidelity | ||
| C1 | TCAAGACAGTGGGCGTCACATACA | CTTCGTGAGACTTACTTTTTTTGTTGC | IN6/809G | 3344 | 62/58 | 5 | 3.0 | −/10 | High Fidelity | |
| C2 | ACACCTGCATGTTCTGATTGG | GCAGGAGACAACTTTGGATCA | 746G/1024T | 879 | 62/58 | 5 | 3.0 | −/10 | High Fidelity | |
| D | AGCAAGGGGAAGCCTCACTCATTC | CCAATGACAATGAGAATG | IN2/IN4 Nested--Ex4 | 419 | 62/57 | 1.5 | - | −/10 | Taq/10X PCR buffer with MgCl2 | |
| KIR2DL4 | A | CACCCACGGTCATCATCC | CCCTTTCSCTGTTGGAGTGT | 28C/IN6 | 5378 | 64/57 | 6 | 2.0 | 2.0/− | High Fidelity |
| A2 | TCCTGGCAGCAGAAGCTGCACC | GGAAAGAGCCGAAGCATC | 5UTR/581G | 2564 | 64/57 | 5 | 2.0 | 2.0/− | High Fidelity | |
| B | CATGTTCTAGGAAACCCTTCT | TGGGCTAAGCAAAGGAGTGT | 666T/3UTR | 5420 | 64/57 | 6 | 2.0 | 2.0/− | High Fidelity | |
| KIR2DL5 | A | ATCTTGTGTTCGGGAGGTTG | TCATAGGGTGAGTCATGGAG | 5UTR/589C | 3274 | 64/62 | 5 | 2.0 | 2.0/− | High Fidelity |
| B | GAGGGGAGGGCCCATGAACC | GGAAGAGCGATCCCCTAAGA | 491C/3UTR | 6193 | 64/62 | 7 | 2.0 | 2.0/− | High Fidelity | |
| A*001+ | CTCCCGTGATGTGGTCAACATGTAAA | TCATAGGGTGAGTCATGGAG | 5UTR/589C | 3109 | 64/62 | 5 | 2.0 | 2.0/− | High Fidelity | |
| B*002+ | CTCCCATGATGTAGTCAACATGTAAG | TCATAGGGTGAGTCATGGAG | 5UTR/589C | 3109 | 64/62 | 5 | 2.0 | 2.0/− | High Fidelity | |
| KIR2DS1 | A | GGCAGCACCATGTCGCTCA | GCATCTGTAGGTCCCTCCA | 10A/576T | 5540 | 64/60 | 7 | 1.5 | 1.0/− | High Fidelity |
| B | TCTCCATCAGTCGCATGAR | GGGTGTCTTGGGCCTCTC | 272R/3UTR | 10227 | 64/60 | 10 | 2.0 | 1.0/− | High Fidelity | |
| KIR2DS2 | A | ATCCTGTGCGCTGCTGAGCTGAG | CACGCTCTCTCCTGCCAA | 5UTR/418T | 5239 | 62/58 | 7 | 1.5 | 2.0/− | High Fidelity |
| B | CTTCTGCACAGAGAGGGGAAGTA | TTATGCGTATGACACCTCCTGAT | 197A/893A | 10253 | 62/58 | 10 | 1.5 | 2.0/− | High Fidelity | |
| KIR2DS3 | A | ATCCTGTGCGCTGCTGAGCTGAG | GCATCTGTAGGTTCCTCCT | 5UTR/576A | 5919 | 64/61 | 7 | 2.0 | −/− | High Fidelity |
| B | GACATGTACCATCTATCCAC | TTATGCGTATGACACCTCCTGATGGTCC | 485C/888G | 8427 | 60/57 | 10 | 2.0 | −/− | High Fidelity | |
| KIR2DS4 | A | CATGTCGCTCATGGTCATCAT | ACACTCTCACCTATGATCACC | 20T/360G | 5122 | 64/58 | 7 | 2.0 | −/− | High Fidelity |
| B | ATCCTGCAATGTTGGTCG | TTATGCGTATGACACCTCCTGAT | 153G/893A | 10299 | 64/58 | 10 | 1.5 | −/− | High Fidelity | |
| C | CGCAGTGACCCTCTGGACATGc | GTGACGGAAACAAGCAGTGGA | 360G/642 T Nested Ex 5 | 1875 | 62/57 | 1.5 | - | −/10 | Taq/10X PCR buffer with MgCl2 | |
| KIR2DS5 | A | CCATCATGATCTTTCTTTCCAGC | CCTCCGTGGGTGGCAGGGT | 35C/563A | 4541 | 62/58 | 5 | 2.0 | −/− | High Fidelity |
| B | CATTGATGGGGTCTCCAAGGG | TTATGCGTATGACACCTCCTGATGGTCC | 248G/888G | 10188 | 62/58 | 10 | 2.0 | −/− | High Fidelity | |
| KIR3DL1 | A | TGTCKRCACCGGCAGCACC | TAGGTCCCTGCAAGGGCAA | 5UTR/560T | 3454 | 60/57 | 5 | 1.5 | 2.0/− | High Fidelity |
| B | CCATCGGTCCCATGATGCT | GACAACTTTGGATCTGGGCTY | 560T/1303Y | 10365 | 60/57 | 11 | - | −/− | Expand Long/Buffer 3 | |
| M | CAARCCCTTCCTGTCTGCCT | GAGAGAGAAGGTTTCTCATATG | 100T/659C | 3265 | 60/57 | 5 | 1.5 | 2.0/− | High Fidelity | |
| KIR3DL2 | A | GTCGTCAGCATGGCGTGC | TGCATCCAAGGCTTCCACC | 30C/IN6 | 8706 | 60/57 | 8 | 1.5 | 2.0/− | High Fidelity |
| A2 | TGTCTGCACCGGCAGCACC | GACCACACGCAGGGCAG | 5UTR/898C | 5421 | 60/57 | 5 | 2.0 | −/10 | High Fidelity | |
| B | TCACATCTCTCCTGTCCCG | GGCTGTTGTCTCCCTAGAAA | IN5/1362T | 7693 | 60/57 | 8 | 1.5 | 2.0/− | High Fidelity | |
| KIR3DL3 | A | TTTCCAGGGTTCTTCTTGCTGG | TGACCCTCAGCACYGCAGT | 49G/799A | 4415 | 62/60 | 5 | 3.0 | 2.0/− | High Fidelity |
| A2 | TGTCTGCACCGGCAGCACC | CCGACAACTCATAGGGTA | 5UTR/605T | 3361 | 62/60 | 5 | 3.0 | −/10 | High Fidelity | |
| B | CCCGGAGCTTGTTTGACATT | AGAAGACAACTTTGGATCTGC | 756T/3UTR | 6569 | 58/54 | 7 | 3.0 | −/− | High Fidelity | |
| KIR3DS1 | A | TGTCKRCACCGGCAGCACC | CTGTGACCATGATCACCAT | 5UTR/A337 | 2116 | 60/57 | 3 | 1.0 | 1.5/− | High Fidelity |
| B | GGCAGAATATTCCAGGAGG | AGAGCGATGCCCTAAGATGA | 235G/3UTR | 12324 | 60/57 | 11 | - | −/− | Expand Long/Buffer 3 | |
b
UTR, untranslated region and/or other 5’ or 3’ noncoding sequences; IN, intron. The designations such as 10T/633G indicates the nucleotide at the annealing site of the 3’ end of the sense/antisense primers. Position 1 is defined as the first nucleotide of the ATG codon in exon 1 according to the IPD/KIR database (http://www.ebi.ac.uk/ipd/kir/). The numbering of KIR2DS4 is based on an allele that does not contain the deletion.
c
Primer sequence is not identical to KIR gene sequence; a substitution was added to avoid the primer from self annealing.