Molecular diagnostic test (application to hemoglobin disorders)

Question 1

What are the different diagnostic methods of hemoglobinopathies?

Answer 1

• Hemoglobinopathies are among the common genetic diseases

1) Protein-based techniques
- Hemoglobin electrophoresis
- High-performance liquid chromatography (HPLC)

2) DNA-Based techniques
- Allele-specific polymerase chain rxn (for known mutations)
- Dot blot hybridization
- Restriction fragment length polymorphism (RFLP)
- DNA-Sequencing (for unknown mutations)

Question 2

Describe the hemoglobin electrophoresis technique

Answer 2

• Identifies the abnormal forms of hemoglobin (variants)
• Electrophoresis is the movement of charged molecules (Hb is -ve) which moves towards the +ve end proportional to their net negative charge by an electric current force
• In electrophoresis, charged molecules are separated, and due to the differences in the charge HbS and other abnormal hemoglobin will migrate differently than the normal Hb
• How to differentiate between HPFH and b-thalassemia since both of them have Hb-F?, in the electrophoresis HPFH will have a band for Hb-A unlike in homozygous thalassemia

Question 3

Describe the high-performance liquid chromatography (HPLC)

Answer 3

• An automated instrument (BioRad Variant) is used for the routine quantification of the Hb variant in a whole-blood
• The program uses a cation exchange to separate and give relative percentages of hemoglobin variants

Question 4

Describe the different HPLC histogram findings

Answer 4

1) Normal adult:
- Hb-A: 95-98%
- Hb-A2: 3.5%
- Hb-F: <1%
- Hb-C: Absent
- Hb-S: Absent

2) Beta thalassemia minor (B0/b, b+/b, “only one allele is affected”)
- Hb-A: 83.6%
- Hb-A2: 6.2%
- Hb-F: 1.1%

3) b-thalassemia major (bo/bo)
- Hb-A: 0%
- Hb-A2: 5-10%
- Hb-F: 90% (gamma genes start overexpressing)

Question 5

Describe the polymerase chain reaction technique

Answer 5

• Amplification of DNA fragments that is located between a pair of primers

we will need:

1) Primer pair: Oligonucleotide DNA sequence (19-26 nucleotide)

2) Taq polymerase

3) Mixture of nucleotides (dNTPs)

4) Mg2+ containing buffer

The PCR cycle is:
1) Denaturation: 96 DEGREES
2) Annealing (binding of the primer): 52-65 DEGREES
3) Extension (DNA synthesis): 72 DEGREES

• For allele-specific PCR, we will be requiring allele-specific primers like (useful for known mutations):

1) Primer for normal allele (Ba “binds to normal sequence only”)
2) Primer for mutant allele (Bs “binds to abnormal sequence only”)

Question 6

Describe the dot blot hybridization

Answer 6

• Detects known mutations

1) DNA denaturation (ssDNA) is required

2) Apply ssDNA onto a nylon membrane (+ve)

3) Incubate the membrane with a biotin-labeled DNA probe complementary to the nucleic acids of interest

4) In subsequent washing steps the non-hybridized probe will be removed

5) Incubate with Avidin reagent for 1 min

6) Image it under the gel documentation system (where it will have missing dots)

Question 7

Describe the restriction fragment length polymorphism (RFLP)

Answer 7

-Used to identify known mutations

• Requires a restriction enzyme, which are endonuclease that recognizes and cuts DNA at specific sites
• Each enzyme has its own specific site on the genome
• Those enzymes are isolated from bacteria

Question 8

How is RFLP used to detect sickle cell anemia?

Answer 8

• In sickle cell anemia the Mst II enzyme restriction site is abolished in the b-globin gene
• The b-globin genes are amplified through PCR which is digested by the Mst II enzyme and separated by aragose gel electrophoresis if the b-globin gene has no mutation
• So when detecting it on the band if a high band was present it means that the enzyme did not digest that gene and thus it is mutated

Question 9

Describe the DNA sequencing technique

Answer 9

-Used for unknown mutations

• We use PCR to amplify the gene before doing the sequence
• It is the alignment of the nucleotide in a correct sequencing
• In the Sanger method (used for 600-700 nucleotides), we use fluorescence dye-labeled (di-deoxyribonucleotide triphosphates “ddNTP”) and we prepare the rxn with 4 different test tubes containing:

1) DNA template
2) A primer
3) dNTPs (dATP, dTTP, dCTP, and dGTP)
4) DNA polymerase
5) one ddNTP (induces chain termination)

• The reaction is then run through a capillary gel to separate the fragments, and passed through a laser detecting ddNTP giving a chromatograph

Question 10

What are the methods used for known mutations?

Answer 10

1) Allele-specific PCR

2) DOT BLOT

Question 11

What is the importance of a chromatograph?

Answer 11

As DNA sequencing is a critical tool to identify errors in the sequence (mutations and SNPs “single nucleotide polymorphism”), in heterozygous you will detect two base peaks and in homozygous you will detect the mutated base peak in the chromatogram graph

Question 12

What is the use of next-generation DNA sequencing (NGS), and what are its advantages over conventional DNA sequencing?

Answer 12

• Perform numerous parallel analyses, analyzing multiple DNA fragments using 10 million tiny wells
• Its advantages are:
• It can screen for both a- and b-thalassemia simultaneously allowing the complete diagnosis of thalassemia
• And a major setback for the conventional DNA sequencing is that it is used for 600-700 nucleotides only