HLA Typing by Real Time PCR

Mohamed Elrefaei, MD, Ph.D.

Clinical Immunology Fellow
 Molecular-Based HLA Typing
• Methods based on sequence detection
– SBT
– PCR-SSP
– PCR-SSOP (forward/reverse)
SBT (Gold Standard)

DNA Isolation

PCR amplification

Sequence reaction

Sequence
purification

Sequence analysis
 PCR-SSP Purified DNA Sample
+
Internal Control Primers
Allele Specific Primers
5’ 3’ 5’ 3’ PCR Amplification Components

3’ 5’ 3’ 5’ 1 2 3 4 5
Allele-Specific Primer Pair #

PCR Amplification

Amplification No Amplification + + + + + Internal Control Amplification

- + + + + No Allele-Specific Amplification

+ - - - - Allele-Specific Amplification

Agarose Gel Electrophoresis

HLA DRB1 L B1 B2 TM C1 C2

HLA-A C1 C2
L a1 a2 a3 TM C3
 PCR-SSOP
DNA Isolation

Generic PCR
amplification

Denaturation of biotinylated
product

Probe Hybridization with

Red fluorescence gctaggtcatcgatgc
group or allele-specific
probes

SAPE binding and

identification
Green fluorescence
 HLA Typing by Real Time PCR
Linkage Biosciences
LinkSēq™ HLA A/B/DR Typing

Technology:
1) Numerous HLA allele sequence-specific primers

2) SYBR® Green fluorescent dye binds to amplified double-stranded DNA product.

3) Results are analyzed by dissociation curves on a real-time PCR instrument. As

double stranded DNA melts, SYBR® Green is released into solution, which causes
an increase in fluorescence.

4) SureTyper™ software identifies the HLA type by comparing the pattern of positive
and negative reactions to known HLA sequences
 HLA Typing by Real Time PCR
Linkage Biosciences
LinkSēq™ HLA A/B/DR Typing

Advantages:
1) No need for separating DNA based on size by gel electrophoresis.
2) Automated pipetting by the Corbet instrument.

Disadvantages:
1) Only HLA A/B/DR typing are currently available.
2) High resolution HLA typing is currently unavailable.
 Results
1) LinkSeq Real-time PCR analysis
HLA A HLA B HLA DRβ
β1
1 (A01, A33) (B15) (DR07, DR13)
2 (A01. A02) (B56, B57) (DR07, DR13)
3 (A23, A80) (B15, B44) (DR08, DR14)
4 (A01, A11) (B38, B40) (DR13)
5 (A02, A29) (B07, B45) (DR01, DR15)

2) LinkSeq PCR + dissociation analysis

HLA A HLA B HLA DRβ
β1
1 (A01, A33) (B15) (DR07, DR13)
2 (A01. A02) (B56, B57) (DR07, DR13)
3 (A23, A80) (B15, B44) (DR08, DR14)
4 (A01, A11) (B38, B40) (DR13)
5 (A02, A29) (B07, B45) (DR01, DR15)
 Results
1) LinkSeq Real-time PCR analysis
HLA A HLA B HLA DRβ
β1
1 (A01, A33) (B15) (DR07, DR13)
2 (A01. A02) (B56, B57) (DR07, DR13)
3 (A23, A80) (B15, B44) (DR08, DR14) 3) SSOP analysis
4 (A01, A11) (B38, B40) (DR13) HLA A HLA B HLA DRβ
β1
5 (A02, A29) (B07, B45) (DR01, DR15) 1 (A01, A33) (B15) (DR07, DR13)
2 (A01. A02) (B56, B57) (DR07, DR13)
3 (A23, A80) (B15, B44) (DR08, DR14)
2) LinkSeq PCR + dissociation analysis 4 (A01, A11) (B38, B40) (DR13)
HLA A HLA B HLA DRβ
β1 5 (A02, A29) (B07, B45) (DR01, DR15)
1 (A01, A33) (B15) (DR07, DR13)
2 (A01. A02) (B56, B57) (DR07, DR13)
3 (A23, A80) (B15, B44) (DR08, DR14)
4 (A01, A11) (B38, B40) (DR13)
5 (A02, A29) (B07, B45) (DR01, DR15)
 Results
LinkSeq Real-time PCR analysis SSP/SSOP analysis
HLA A HLA B HLA DRβ
β1 HLA A HLA B HLA DRβ
β1
1 A24 B07 DR01 1 A24 B07 DR01
2 A01, A29 B08, B45 DR04, DR13 2 A01, A29 B08, B45 DR04, DR13
3 A34, A74 B15, B58 DR07, DR15 3 A34, A74 B15, B58 DR07, DR15
4 A01, A29 B27, B57 DR01, DR07 4 A01, A29 B27, B57 DR01, DR07
5 A01, A02 B40, B57 DR07, DR15 5 A01, A02 B40, B57 DR07, DR15
6 A24, A34 B40 DR01, DR11 6 A24, A34 B40 DR01, DR11
7 A01, A02 B08, B44 DR04 7 A01, A02 B08, B44 DR04
8 A11, A24 B38, B51 DR09, DR16 8 A11, A24 B38, B51 DR09, DR16
9 A01, A24 B40, B52 DR11, DR15 9 A01, A24 B40, B52 DR11, DR15
10 A02, A66 B35, B47 DR08, DR11 10 A02, A66 B35, B47 DR08, DR11
11 A02 B40, B44 DR07, DR14 11 A02 B40, B44 DR07, DR14
12 A02 B40 DR09 12 A02 B40 DR09
13 A30, A31 B18, B40 DR11 13 A30, A31 B18, B40 DR11
14 A02, A23 B42, B81 DR03, DR08 14 A02, A23 B42, B81 DR03, DR08
15 A02, A30 B18, B35 DR03, DR11 15 A02, A30 B18, B35 DR03, DR11
16 A01, A11 B38, B40 DR13 16 A01, A11 B38, B40 DR13
17 A02, A29 B07, B45 DR01, DR15 17 A02, A29 B07, B45 DR01, DR15
18 A24, A34 B38, B48 DR14, DR15 18 A24, A34 B38, B48 DR14, DR15
19 A01, A02 B35, B37 DR10, DR15 19 A01, A02 B35, B37 DR10, DR15
20 A02, A23 B42, B81 DR03, DR08 20 A02, A23 B42, B81 DR03, DR08
 Acknowledgements

UNC HLA lab

John Crawford
Rosanne Petraroia
Hector Jimenez
Nicole Furmage
Tricia Crawford
Holly Brown
Jennifer Haugle
Andrea Parkinson

Dr. John Schmitz