1. DNA Analysis of the HLA Gene Complex
Focus: HLA gene complex - major functions of immune system - direct role in organ transplants Goal: Determine student HLA Genotypes using modern biotechnology instruments and technology.

2. Organ Transplants
Pre-transplant: Determine who will accept a transplanted organ / tissue (bone marrow) and who will reject it.
Post-transplant: Once a patient receives a transplant, how to identify those that may be rejecting their organ / tissue (bone marrow).
A significant driver: mismatched organs and poor transplant outcomes cost the US Healthcare System $1.5 billion annually.

3. How do you match organs and patients?
Patient and organ must have same HLA genotype
HLA: Human Leukocyte Antigen
HLA genes control our immune system responses
If the patient’s HLA system sees the organ as a foreign invader, the body will attack it and cause organ rejection.

4. HLA: Self vs. Non-Self + = + =
HLA proteins are the body’s “surveillance system”
The “HLA System” discriminates between “self” and “non-self”
“Self” is OKAY, but “Non-Self” is NOT OKAY and the body tries to eliminate it
“Self”
Immune System Cells
Response
Proteins on your own cells
MHC +
T cell or
B cell =
No Attack
“Non-Self”
Bacteria & Viruses
MHC
T cell or
B cell + =
Attack!
Proteins on cells that are NOT your own
(Example: transplanted tissue or organ)

5. HLA Gene Complex
All HLA proteins are coded by genes located on short arm of chromosome 6
Spans about 4 megabases
Contains more than 200 loci (genes)

6. HLA Gene Complex
Highest polymorphism of any human genetic system (more than 2,400 variants/alleles of these genes)
Associated with more diseases than any other genetic region
Divided into Class I (loci A, B and C), Class II (DR, DQ..) and Class III

7. HLA Genes and Alleles
Within a gene locus, there are different alleles creating different proteins on the surface of cells.
For example, HLA-A2 and HLA-A68 class I molecules differ from one by 13 amino acids
These differences give rise to dramatic differences in protein structure and shape.
The alleles can be expressed co-dominantly

8. HLA Allele Discoveries

9. Current HLA Allele List

10. Allele Names and Numbers
Indicates
HLA
the HLA region and prefix for an HLA gene
HLA-DRB1
a particular HLA locus i.e. DRB1
HLA-DRB1*13
a group of alleles which encode the DR13 antigen
HLA-DRB1*1301
a specific HLA allele
HLA-DRB1*1301N
a null allele
HLA-DRB1*130102
an allele which differs by a synonymous mutation

11. How do you find the HLA genotype?
SSO Probes
Sequence Specific Oligonucleotide Probes:
Small segments of single stranded DNA ready to bond or “hybridize” with a second matching strand of DNA.
Only correctly matching DNA probes hybridize with a DNA sample, so we can see which probes are positive matches to find a genotype.

12. SSO Genotype Analysis
SSO probes are short segments of single stranded DNA. Each probe has a unique DNA sequence.
Matching DNA will hybridize on the probe.
Then, chemicals markers are added so we can detect which DNA matches.
Detection
Hybridization
We can see the detection chemicals on the probes, and we can find the genotype.

13. SSO Probe Sample Data Dark spots show positive matches on SSO probes.
Weak or blank spots show no match.

14. SSO Systems: RELI vs. Prodigy
Probes on membrane Strips
Limited # of Probes
More hands on time
Larger quantities of reagents
External Scanner for imaging
Low to medium resolution
Prodigy
Probes on chips
More than 500 Probes/chip
Fully automated
Smaller quantities of reagents
Internal camera for imaging
Medium to high resolution

15. Inside the Prodigy Instrument

16. RELI Manual Assay We can perform the procedure manually
without the RELI or Prodigy instrument and
get the exact same results!

17. SSO Manual Assay
All of these molecules can be attached to the SSO probes by simply applying the correct chemical reagents.
Detection
Hybridization

18. RELI Strips
The RELI system creates paper strips with dark bands showing positive DNA matches to the SSO probes.
Each student will add their own DNA sample to their own RELI strip and read the positive matches to find the HLA genotype.

19. Weak positive = Matching DNA
RELI Strip Analysis
There are many probes on each RELI strip, but only certain ones will show a positive match to your DNA sample.
Start
Stop
Control Probes
Weak positive = Matching DNA
Strong Positive = Matching DNA
Negative = No matches

20. RELI Strip Analysis
We will use an Overlay and a Scoresheet to analyze and record all positive and negative matches on your RELI strip.