High Resolution HLA Analysis in Situations of STR Marker Depletion

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The Human Leukocyte Antigen (HLA) molecules are cell-surface proteins found in most cells in the human body (except red blood cells). HLA has one of the most
polymorphic gene systems, comprising over 1000 alleles in the Class I (A, B and C) and Class II (DR, DQ and DP). In fact, HLA typing has been used since 1970s to determine paternity in the USA and worldwide.Therefore, HLA typing method is an excellent addition for DNA testing.

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High Resolution HLA Analysis in Situations of STR Marker Depletion

  1. 1. High Resolution HLA Analysis in Situations of STR Marker Depletion Kien Tjhen, M.S., Anwu Zhou, Ph.D., Jessica Castro, M.S., Miguel Castro, Ph.D. DNA Identity Testing Center, Bio-Synthesis, Inc., Lewisville, Texas 75057 Table 3: Example results of HLA-DRB1 LocusAbstract Materials Tables System Obligate allele AF* PI FormulaThe Human Leukocyte Antigen (HLA) molecules are 1. Buccal swab samples (AF and CH) from paternity case Table 1: 21 STR markers DRB1 0407 0.06428 7.778 1/2acell-surface proteins found in most cells in the human used for this experiment 1 D8S1179 DRB1 1504 0.00025 2000 1/2abody (except red blood cells). HLA has one of the most 2. Chelex 2 D21S11 *Allele frequency (AF) data is based on Hispanic populations conducted by Nationalpolymorphic gene systems, comprising over 1000 3. Protease K 3 D7S820 Marrow Donor Program (2007-11-20)alleles in the Class I (A, B and C) and Class II (DR, DQ 4. Centricon (10kD cutoff ) 4 CSF1PO When the CPI and PP values are low as shown in table 2,and DP). In fact, HLA typing has been used since 1970s 5. High resolution HLA -DRB1 kit (cat# RSSOH2B1) from 5 D3S1358 HLA typing experiment was used to check additionalto determine paternity in the USA and worldwide. One Lambda, Inc. 6 THO1 7 D13S17 markers. For example, the allele 0407 (see table 3), which isTherefore, HLA typing method is an excellent additionfor DNA testing. Methods 8 D16S539 a common allele, was taken into consideration, the CPI and 9 D2S1338 PP increased to 460.796 and 99.78% respectively. However,Advantages of HLA DNA Extraction and Luminex Procedures 10 D19S433 the allele 1504, which is rare, was taken into consideration, Chelex Extraction: 11 VWA the CPI and PP increased significantly to 118490.2 andMarker Exhaustion: 1. Prepare a 5% suspension of Chelex 100 resin, then put 12 TPOX 99.9992%.When using the 15-marker Identifiler kit (excluding 200 ul into a tube. 13 D18S51Amelogenin), the CPI (Combined Paternity Index) may 2. Add 750 ul of sterile water. 14 D5S818 Conclusion 15 FGAnot reach 100 and PP (Probability of Paternity) may not 3. Add 100 ul of protease K solution (20 mgs/ml). HLA typing is an excellent addition for STR profiling. One 16 F13A01be greater than 99% (see table 2). Hence, most 4. Add 20 ul of whole blood. HLA locus, such as HLA-DRB1, is sufficient to increase the 17 F13Bscientists utilize additional STRs to increase the CPI 5. Vortex tube for 5 sec, then incubate at 56°C for 20 min. 18 FESFPS value of CPI and PP significantly.and PP. Even though additional markers (see table 1) 6. Vortex tube for 5 sec, then incubate at 100°C for 8 min. 19 LPL Advantages:can be used, the result may not be sufficient, especially 7. Vortex tube for 5 sec, then centrifuge at 10k rpm for 3 20 Penta Dfor common alleles. Therefore, HLA is very useful in 1. HLA typing results give a unique allele with known min. 21 Penta EDNA testing. One locus of HLA marker could frequency that could increase CPI and PP significantly. 8. Transfer 600 ul of the supernatant to centricon (10ksignificantly increase the value of CPI and PP. Table 2: Example results of 15 STR markers 2. HLA typing uses the same DNA extracts for STR typing, cutoff ), then centrifuge at 2500 rpm for 20 min. such as DNA from buccal swab, blood, etc.Mutations: 9. Transfer the supernatant to new tube . Child Alleged Father 3. Turn around time is approximately 4 hours.In cases where there are mutations, HLA analysis will 10. Read the concentration. The amount needed for System Allele Allele Allele Allele PI 4. Commercial Luminex kit available.help to confirm paternity for inconclusive cases. Luminex analysis is 80-100 ngs. D8S1179 14 14 15 1.5002 11. DNA is ready for PCR.Disease Association: D21S11 29 31 29 32.2 1.3165There are a number of immune related diseases that Luminex instrument for HLA analysis: D7S820 8 10 10 11 0.7721correlate with specific HLA alleles. The following protocols are available at CSF1PO 11 13 8 11 1.2207Extended Analysis: www.onelambda.com: D3S1358 15 16 16 18 0.8141 TH01 7 8 7 1.1351In cases where a higher CPI is needed, instead of using 1. PCR master mix preparation D13S317 12 12 13 1.0348hard-to-find places that perform VNTR analysis, HLA 2. PCR condition/parameters D16S539 9 10 9 11 1.2588analysis can increase the CPI values of any given case 3. Denaturization and neutralization D2S1338 17 19 19 23 1.7668significantly. 4. Hybridization and washing D19S433 13 14 13 13.2 0.8381 5. Labeling vWA 16 19 15 19 3.4626 6. Data Acquisition and Luminex machine preparation fd TPOX 8 11 8 11 1.7902 7. Data analysis D18S51 16 17 17 20 1.5253 -Identifiler® (ID) kit, Applied D5S818 12 11 12 1.4061 Biosystems (cat# 4322288) FGA 22 24 18.2 24 1.3434 -Thermocycler, GeneAmp CPI: 59.2451 PP: 98.3401% 9700, Applied Biosystems -ABI 310 Genetic Analyzer www.biosyn.com Service across Genomics, Proteomics and Cell Biology Toll Free: 800.227.0627 | 972.420.8505 Mail: info@biosyn.com

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