Beta-Thalassemia PCR for Sequencing

Introduction

Beta-thalassemia PCR is one of diagnostic laboratory methods used to identify the specific mutation involved in beta-thalassemia. Beta-thalassemia is a group of inherited blood disorders characterized by a reduced or absent production of beta-globin, a protein chain that makes up hemoglobin, the oxygen-carrying protein in red blood cells. This deficiency leads to the formation of abnormally small, pale red blood cells (anemia), which can cause a range of symptoms, including fatigue, weakness, pale skin, and shortness of breath.

There are two main types of beta-thalassemia: beta-thalassemia major and beta-thalassemia minor. Beta-thalassemia major, also known as Cooley’s anemia, is the most severe form of the disorder. It is caused by mutations in both copies of the beta-globin gene and typically manifests in early childhood. Symptoms of beta-thalassemia major can be severe and may include frequent blood transfusions, bone deformities, and heart problems.

Laboratory investigations play a crucial role in diagnosing and monitoring beta-thalassemia. These tests can help determine the type and severity of the disorder, assess the effectiveness of treatment, and identify potential complications.

DNA analysis, such as gene sequencing, can identify the specific mutations in the beta-globin gene that cause beta-thalassemia. This information can be used for genetic counselling and prenatal diagnosis.

The purpose of this protocol is to amplify the whole β-globin gene as PCR products so that they can be sent for direct sequencing to identify unknown beta-thalassemia mutations. Three segments of amplicons encompassing the entire 1.6 kb of β-globin gene including the 5’ and 3’ of the untranslated region were expected to cover all possible mutations occurring in the β-globin gene.

Schematic diagram showing the three segments of the whole β-globin gene PCR-amplified for direct genomic sequencing.  Three primer pairs were indicated as forward and backward arrows. Set A, B and C have the amplified sizes of 916, 902 and 855 base pairs, respectively. The relative positions were not shown in actual scale. 

Principle of PCR

Polymerase chain reaction (PCR) is a technique used to amplify small segments of DNA into millions or billions of copies. It is a powerful tool in molecular biology and is used in a wide range of applications, including:

• Research: PCR is used to study DNA from a variety of sources, including viruses, bacteria, plants, and animals. It can be used to identify new genes, study gene expression, and detect mutations.
• Diagnostics: PCR is used to diagnose a variety of diseases, including infectious diseases, genetic disorders, and cancer.
• Forensic science: PCR is used to identify individuals from DNA samples found at crime scenes.

PCR is based on the ability of DNA polymerase to synthesize new DNA strands complementary to a given template strand. The reaction is carried out in a series of cycles, each of which consists of the following steps:

1. Denaturation: The double-stranded DNA template is heated to denature it, separating the two strands.
2. Annealing: Short oligonucleotide primers, which are complementary to the ends of the target DNA sequence, are added to the reaction mixture. The primers bind to the template DNA at the desired amplification sites.
3. Extension: DNA polymerase is added to the reaction mixture and synthesizes new DNA strands complementary to the template DNA and primers.

The PCR cycle is repeated 20-40 times, resulting in an exponential amplification of the target DNA sequence. At the end of the PCR reaction, the amplified DNA can be analyzed or used in other downstream applications.

PCR is a highly sensitive and specific technique. It can be used to amplify DNA from even a few cells or from DNA that is degraded or damaged. PCR is also a relatively fast and easy technique to perform.

Here is a more detailed explanation of the steps involved in PCR:

Denaturation

Denaturation is the process of separating the two strands of double-stranded DNA. This is done by heating the DNA to a high temperature, typically 95 degrees Celsius. The high temperature breaks the hydrogen bonds that hold the two strands of DNA together.

Annealing

Annealing is the process of binding the primers to the template DNA. Primers are short oligonucleotide sequences that are complementary to the ends of the target DNA sequence. Primers are important because they help to ensure that the DNA polymerase only amplifies the desired DNA sequence.

Extension

Extension is the process of synthesizing new DNA strands complementary to the template DNA and primers. This is done by adding DNA polymerase to the reaction mixture. DNA polymerase is an enzyme that can synthesize new DNA strands using a template strand and a primer.

The PCR cycle is repeated 20-30 times, resulting in an exponential amplification of the target DNA sequence. At the end of the PCR reaction, the amplified DNA can be analyzed or used in other downstream applications.

PCR is a powerful and versatile technique that is used in a wide range of applications. It is a valuable tool for researchers, clinicians, and forensic scientists.

Materials

• PCR master mix kit
• Primers 
• Pipet tips with aerosol barrier 
• Pipettes
• Vortex
• Nuclease-free water
• Genomic DNA
• 1.5 ml microfuge tubes
• 0.2 ml PCR tube/strips
• Thermalcycler
• Gloves
• 70% alcohol (ethanol/isopropanol) spray

Protocol

Table 1. Primer sequences for β-globin gene sequencing.

Primer ID	Primer sequences (5’ to 3’)	Location: bases (Appendix 3.3c)	Ta(°C)	Amplicon size (bp)
		HUMHBB U01317:
βA – F	CGA TCT TCA ATA TGC TTA CCA A	61830-61851	60	916
βA – R	CAT TCG TCT GTT TCC CAT TCT A	62745-62725
βB – F	GCA CGT GGA TCC TGA GAA CT	62607-62626	68	902
βB – R	CAC ACA GAC CAG CAC GTT G	63508-63490
βC – F	GCT AAT CAT GTT CAT ACC TCT T	63445-63466	62	855
βC – R	CAG ATT CCG GGT CAC TGT G	64299-64281

*The concentration of each primer pair is 4 pmol. 

1. Wipe down the PCR work bench and pipettes with alcohol spray and turn on UV light for 10 minutes before starting any work.
2. Thaw samples and reagents by hand, agitate and spin down. All components should be mixed and spun down prior to pipetting. 
3. Next, prepare a master mix for the PCR reaction in a 1.5 mL microcentrifuge tube on ice as shown in Table 2. 

Table 2. Preparation of Master Mix for PCR reaction.

Component	Each reaction(50 μL)	Final concentration
PCR Master mix*	25 µL	1×
Primer mixes (Forward and reverse) 10mM stock for each primers	4 µL	0.4 µM for each primer
Nuclease free water	18 µL	–
Template DNA (can be added later e.g. Step 4)	3.0 µL	~300 ng
Total	50 µL

4. Mix master mix gently and spin down using a microcentrifuge.
5. Add 47 µL of master mix to each respective PCR tube with pre-loaded 3 µL of template DNA on ice.
6. Set the cycling conditions or use the Beta Sequence A/B/C (depending on the set being used) programme in the PCR Thermal Cycler machine as shown in Table 3 and start the run. 

Table 3: Thermocycling conditions

Step	Temperature (°C)	Time (minute(s))
Initial Denaturation	95°C	10 minutes
Cycling 30 cycles	95°C	1 minute
	Ta °C	1 minute
	72°C	1 minute 30 seconds
Final Extension	72°C	10 minutes

7. Proceed to gel electrophoresis.
8. The PCR products should be purified before using it for sequencing.

Interpretation

This gel electrophoresis image reveals the distinct bands representing successful amplification of the entire beta globin gene, which will be used for downstream sequencing protocols after PCR clean up protocol.

Disclaimer: This protocol is intended for informational purposes only and may need to be modified depending on the specific laboratory procedures and patient circumstances. Always consult with a qualified healthcare professional for guidance. See additional information.

References

1. Lim WF, Muniandi L, George E, Sathar J, Teh LK, Gan GG, Lai MI. α-Haemoglobin stabilising protein expression is influenced by mean cell haemoglobin and HbF levels in HbE/β-thalassaemia individuals. Blood Cells Mol Dis. 2012 Jan 15;48(1):17-21. doi: 10.1016/j.bcmd.2011.10.002. Epub 2011 Nov 12. PMID: 22079025.