high resolution hla analysis in situations of str marker depletion

Abstract

Advantages of HLA

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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.

1. Buccal swab samples (AF and CH) from paternity case used for this experiment

2. Chelex3. Protease K4. Centricon (10kD cutoff)5. High resolution HLA -DRB1 kit (cat# RSSOH2B1) from

One Lambda, Inc.

Marker Exhaustion:When using the 15-marker Identifiler kit (excluding Amelogenin), the CPI (Combined Paternity Index) may not reach 100 and PP (Probability of Paternity) may not be greater than 99% (see table 2). Hence, most scientists utilize additional STRs to increase the CPI and PP. Even though additional markers (see table 1) can be used, the result may not be sufficient, especially for common alleles. Therefore, HLA is very useful in DNA testing. One locus of HLA marker could significantly increase the value of CPI and PP.

Mutations:In cases where there are mutations, HLA analysis will help to confirm paternity for inconclusive cases.

Disease Association:There are a number of immune related diseases that correlate with specific HLA alleles.

Extended Analysis:In cases where a higher CPI is needed, instead of using hard-to-find places that perform VNTR analysis, HLA analysis can increase the CPI values of any given case significantly.

TablesMaterials

1. Prepare a 5% suspension of Chelex 100 resin, then put 200 ul into a tube.

2. Add 750 ul of sterile water.3. Add 100 ul of protease K solution (20 mgs/ml).4. Add 20 ul of whole blood.5. Vortex tube for 5 sec, then incubate at 56°C for 20 min.6. Vortex tube for 5 sec, then incubate at 100°C for 8 min.7. Vortex tube for 5 sec, then centrifuge at 10k rpm for 3

min.8. Transfer 600 ul of the supernatant to centricon (10k

cutoff), then centrifuge at 2500 rpm for 20 min.9. Transfer the supernatant to new tube .10. Read the concentration. The amount needed for

Luminex analysis is 80-100 ngs. 11. DNA is ready for PCR.

1. PCR master mix preparation2. PCR condition/parameters3. Denaturization and neutralization4. Hybridization and washing5. Labeling6. Data Acquisition and Luminex machine preparation7. Data analysis

DNA Extraction and Luminex ProceduresChelex Extraction:

Luminex instrument for HLA analysis:The following protocols are available at www.onelambda.com:

Methods

Table 1: 21 STR markers

Table 2: Example results of 15 STR markers

High Resolution HLA Analysis in Situations of STR Marker DepletionKien 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

-Identifiler® (ID) kit, Applied Biosystems (cat# 4322288)

-Thermocycler, GeneAmp 9700, Applied Biosystems

-ABI 310 Genetic Analyzer

1. HLA typing results give a unique allele with known frequency that could increase CPI and PP significantly.

2. HLA typing uses the same DNA extracts for STR typing, such as DNA from buccal swab, blood, etc.

3. Turn around time is approximately 4 hours.4. Commercial Luminex kit available.

ConclusionHLA typing is an excellent addition for STR profiling. One HLA locus, such as HLA-DRB1, is sufficient to increase the value of CPI and PP significantly.

When the CPI and PP values are low as shown in table 2, HLA typing experiment was used to check additional markers. For example, the allele 0407 (see table 3), which is a common allele, was taken into consideration, the CPI and PP increased to 460.796 and 99.78% respectively. However, the allele 1504, which is rare, was taken into consideration, the CPI and PP increased significantly to 118490.2 and 99.9992%.

Table 3: Example results of HLA-DRB1 Locus

Advantages:

*Allele frequency (AF) data is based on Hispanic populations conducted by National Marrow Donor Program (2007-11-20)

System Obligate allele AF* PI Formula

DRB1 0407 0.06428 7.778 1/2a

DRB1 1504 0.00025 2000 1/2a1 D8S1179

2 D21S11

3 D7S820

4 CSF1PO

5 D3S1358

6 THO1

7 D13S17

8 D16S539

9 D2S1338

10 D19S433

11 VWA

12 TPOX

13 D18S51

14 D5S818

15 FGA

16 F13A01

17 F13B

18 FESFPS

19 LPL

20 Penta D

21 Penta E

CPI: 59.2451 PP: 98.3401%

Child Alleged Father

System Allele Allele Allele Allele PI

D8S1179 14 14 15 1.5002

D21S11 29 31 29 32.2 1.3165

D7S820 8 10 10 11 0.7721

CSF1PO 11 13 8 11 1.2207

D3S1358 15 16 16 18 0.8141

TH01 7 8 7 1.1351

D13S317 12 12 13 1.0348

D16S539 9 10 9 11 1.2588

D2S1338 17 19 19 23 1.7668

D19S433 13 14 13 13.2 0.8381

vWA 16 19 15 19 3.4626

TPOX 8 11 8 11 1.7902

D18S51 16 17 17 20 1.5253

D5S818 12 11 12 1.4061

FGA 22 24 18.2 24 1.3434

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