# Presentation: 185697

## New Primer Sets for Y Chromosome and CODIS STR Loci

**Christian M. Ruitberg and John M. Butler**
**Biotechnology Division, National Institute of Standards and Technology, 100 Bureau Drive MS 8311, Gaithersburg, MD 20899-8311**

**Methods and Materials**
**Small STR Amplicon Work**
**PCR**: 10 uL volume; 1 U TaqGold; AmpFlSTR® PCR mix; 12 pmol each primer; 5-25 ng DNA template
**Thermal Cycling**: 95 oC-10 min; 25 cycles: 94 oC-1 min, 59 oC-1 min, 72 oC-1 min; 45 min 60 oC
**Thermal Cycler**: PE9700 (9600 emulation)
**ABI 310**: Filter F, POP4, 47 cm uncoated capillary, injection: 5 s @ 15 kV; separation: 15 kV
**Dye Matrix**: FAM, JOE, NED, ROX
**Y STR Multiplex Work**
**PCR**: 20 uL volume; 2 U TaqGold; 2 mM MgCl2; 5-20 pmol each primer; 5-25 ng DNA template
**Thermal Cycling**: 95 oC-10 min; 25 cycles: 94 oC-1 min, 55 oC-1 min, 72 oC-1 min; 45 min 60 oC
**Thermal Cycler**: PE9700 (9600 emulation)
**ABI 310**: Filter C, POP4, 47 cm uncoated capillary, injection: 5 s @ 15 kV; separation: 15 kV
**Dye Matrix**: FAM, TET, HEX, ROX

**New Primer Sets for Several CODIS STR Loci**

| Locus | Current Kits | New Primer Sets | Primer Sequences* |
| --- | --- | --- | --- |
| CSF1PO | 282-318 bp | 161-197 bp; 86-122 bp | 5’-JOE-ACAGTAACTGCCTTCATAGATAG-3’
5’-GTGTCAGACCCTGTTCTAAGTA-3’  |
| TH01 | 168-188 bp | 55-98 bp | 5’- JOE- CCTGTTCCTCCCTTATTTCCC-3’
5’-GGGAACACAGACTCCATGGTG-3’  |
| TPOX | 217-241 bp | 69-101 bp | 5’- JOE-CTTAGGGAACCCTCACTGAATG-3’
5’-GTCCTTGTCAGCGTTTATTTGC-3’  |
| D7S820 | 257-293 bp | 66-98 bp | 5’- JOE-TGTCATAGTTTAGAACGAACTAAC-3’
5’-AAAAACTATCAATCTGTCTATCTATC-3’  |
| D16S539 | 233-273 bp | 81-121 bp | 5’-JOE-ATACAGACAGACAGACAGGTG-3’ 
5’-GCATGTATCTATCATCCATCTCT-3’ |
| Penta D | 376-449 bp | 96-169 bp | 5’- JOE-GAGCAAGACACCATCTCAAGAAA-3’
5’-GAGAAATTTTACATTTATGTTTATGATTCTCTTT-3’ |

| Locus | Previously Published | New Primer Sets | Primer Sequences |
| --- | --- | --- | --- |
| Y19 | 178-210 bp | 178-210 bp | Forward (20 pmol): FAM-CTACTGAGTTTCTGTTATAGT
Reverse (20 pmol): ATGGCATGTAGTGAGGACA |
| Y391 | 275-295 bp | 155-175 bp | Forward (10 pmol): TET-CTATTCATTCAATCATACACCCATAT
Reverse (10 pmol): ACATAGCCAAATATCTCCTGGG |
| Y392 | 236-263 bp | 95-122 bp | Forward (20 pmol): HEX-AAAAGCCAAGAAGGAAAACAAA
Reverse (20 pmol): AAACCTACCAATCCCATTCCTT |
| Y435 | 210-228 bp | 152-160 bp | Forward (5 pmol): GGGTTGTCCAGAGAAACAGC
Reverse (5 pmol): FAM-CCCCCTCCTCTCGTCTATCT |
| Y436 | 128-143 bp | 85-100 bp | Forward (10 pmol): CCAGGAGAGCACACACAAAA
Reverse (10 pmol): FAM-ACGAGCTGCGTTAGAGGTGA |
| Y437 | 186-202 bp | 186-202 bp | Forward (10 pmol): GACTATGGGCGTGAGTGCAT
Reverse (10 pmol): HEX-AGACCCTGTCATTCACAGATGA |
| Y438 | 203-233 bp | 135-165 bp | Forward (10 pmol): HEX-TGGGGAATAGTTGAACGGTAA
Reverse (10 pmol): GGAGGTTGTGGTGAGTCGAG |
| Y439 | 238-258 bp | 118-138 bp | Forward (10 pmol): FAM-ACATAGGTGGAGACAGATAGATGAT
Reverse (10 pmol): GCCTGGCTTGGAATTCTTTT |
| A7.1 | 161-181 bp | 99-119 bp | Forward (10 pmol): GAGGAATCTGACACCTCTGACA
Reverse (10 pmol): TET-TCCATATCATCTATCCTCTGCCTA |
| H4 | 362-370 bp | 131-139 bp | Forward (20 pmol): TET-ATGCTGAGGAGAATTTCCAA
Reverse (20 pmol): CTATTCATCCATCTAATCTATCCATT |

**Y STR 10plex: Product Sizes and Primer Sequences**

| Sample
# | Y439 | Y19 | A7.1 | H4 | Y391 | Y392 | Y437 | Y438 | Y435 | Y436 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | C | A | B | B | B | B | B | B | A | A |
| 2 | A | C | A | B | A | A | A | A | A | A |
| 3 | D | A | B | B | B | B | A | B | A | A |
| 4 | B | B | B | B | A | A | B | A | A | A |
| 5 | C | C | A | B | A | A | A | A | A | A |
| 6 | D | C | C | A | A | A | A | A | A | A |
| 7 | D | B | B | A | A | A | A | A | A | A |
| 8 | C | A | A | B | B | B | B | B | A | A |
| 9 | C | A | B | B | B | B | A | B | A | A |
| 10 | D | A | A | A | A | A | A | A | A | A |