Sickle Cell Lab

What is the full name for PCR?

Polymerase Chain Reaction.

What materials are required to complete Part 1 (PCR)?

Micropipette and disposable tips, patient DNA samples (J, C, S, M), Master mix (polymerase, dNTPs, buffer, MgCl₂), primers specific to the haemoglobin gene, miniPCR thermocycler device and software, PCR tubes.

What is inside Sample J, C, S, M and what is their purpose?

Each sample contains genomic DNA from a specific family member (Jacqueline, Cory, Samuel, or Marie) to serve as template DNA for amplifying the target haemoglobin gene sequence.

What are the contents and role of the PCR Mastermix?

Contents: Taq polymerase, dNTPs (A, T, C, G), reaction buffer, MgCl₂. Role: Provides the enzyme, building blocks, and chemical environment needed to synthesize new DNA strands.

What are primers and what is their role in PCR?

Short, single-stranded synthetic DNA sequences that bind specifically to complementary sequences, providing a 3'-OH starting site for DNA polymerase.

What happens during the Denaturation step of PCR?

The reaction is heated to ~95°C to break hydrogen bonds between complementary bases, separating double-stranded template DNA into two single strands.

What happens during the Annealing step of PCR?

The reaction is cooled to ~50–60°C, allowing single-stranded DNA primers to hybridize (bind) to complementary sequences on the single-stranded template.

What happens during the Extension step of PCR?

The reaction is heated to ~72°C where Taq DNA polymerase binds to primers and synthesizes new complementary DNA strands by adding dNTPs in the 5' to 3' direction.

Starting with 1 template strand, how many target DNA copies exist after 1, 10, and 30 PCR cycles?

1 cycle: 2 copies; 10 cycles: 1,024 copies; 30 cycles: 1,073,741,824 copies.

What restriction sequence does DdeI recognize and where does it cut?

DdeI recognizes 5'-C↓TNAG-3' and cuts between the C and T on the 5' side.

How does DdeI distinguish between the normal allele (HbA) and sickle cell allele (HbS)?

HbA contains the intact recognition site (CTGAG) and is cut into 250 bp and 150 bp fragments. HbS has a point mutation (CTGTG) that destroys the site, leaving an uncut 400 bp fragment.

What materials are required for gel electrophoresis and what are their functions?

Agarose gel (separates fragments by size), 1X TBE buffer (conducts current/pH stability), 100 bp DNA ladder (size marker), digested DNA samples, electrophoresis unit (electric field), blue light visualizer (fluoresces DNA stain).

What happens to DNA particles during gel electrophoresis?

Negatively charged DNA fragments migrate through the porous agarose gel matrix away from the negative cathode toward the positive anode.

How does gel electrophoresis separate and visualize DNA fragment lengths?

Shorter DNA fragments move faster through gel pores while longer fragments move slower. Comparing migration distances against a DNA ladder reveals fragment size in base pairs.

What is a DNA ladder and why is it used?

A mixture of pre-cut DNA fragments of known base pair sizes loaded alongside test samples to serve as a ruler for measuring unknown sample band lengths.

What was Jacqueline's gel result and diagnosis?

Result: 3 bands (400 bp, 250 bp, 150 bp). Diagnosis: Sickle Cell Trait / Carrier (HbA/HbS).

What was Cory's gel result and diagnosis?

Result: 3 bands (400 bp, 250 bp, 150 bp). Diagnosis: Sickle Cell Trait / Carrier (HbA/HbS).

What was Samuel's gel result and diagnosis?

Result: 2 bands (250 bp, 150 bp). Diagnosis: Unaffected / Normal Hemoglobin (HbA/HbA).

What was Marie's gel result and diagnosis?

Result: 1 single band (400 bp). Diagnosis: Sickle Cell Disease (HbS/HbS).

What phenotypic traits are associated with the HbA/HbA genotype?

Normal hemoglobin phenotype, normal biconcave red blood cells, no sickle symptoms, fully susceptible to severe malaria.

What phenotypic traits are associated with the HbA/HbS genotype?

Sickle Cell Trait / Asymptomatic carrier, normal RBC function under normal conditions, partial protection/resistance against severe malaria (Heterozygote Advantage).

What phenotypic traits are associated with the HbS/HbS genotype?

Sickle Cell Disease, abnormal HbS polymerizes deforming RBCs into rigid sickle shapes, causes chronic anemia, pain crises, organ damage, and reduced lifespan.

Why is carrying the sickle cell trait (HbA/HbS) advantageous in areas with high malaria prevalence?

Infected red blood cells sickle prematurely and are destroyed by the spleen, removing the Plasmodium parasite before it can multiply and cause severe malaria.

Which geographic region of Africa shows the highest frequency of the HbS allele?

Sub-Saharan Africa, specifically Central and Western equatorial Africa.

What is the relationship between malaria prevalence and HbS allele frequency?

Direct geographic correlation: regions with high malaria endemicity have high HbS frequencies due to natural selection favoring carriers.

Why is sickle cell disease a significant concern in African American populations?

The transatlantic slave trade forcibly relocated populations from West Africa (where HbS was high due to malaria selection), introducing the allele into the American gene pool.

In endemic malaria regions, which is more disadvantageous: having sickle cell disease (HbS/HbS) or normal hemoglobin (HbA/HbA)?

Sickle cell disease (HbS/HbS), due to high early childhood mortality (>90% before age 5) from severe blood crises compared to malaria risk alone.

Which allele (HbA or HbS) remains more common in populations where malaria is present?

The HbA allele (>70–80%), because strong negative selection against HbS/HbS homozygotes prevents HbS from becoming the majority allele.

If two carrier parents (HbA/HbS) have a child, what is the probability the child will have sickle cell disease?

25% (1 in 4 chance) for HbS/HbS.

Can an individual with sickle cell disease (HbS/HbS) and a non-carrier (HbA/HbA) have a child with sickle cell disease?

No. 100% of offspring will be heterozygous carriers (HbA/HbS).

What pedigree evidence proves sickle cell disease is an autosomal recessive trait?

Two unaffected carrier parents (Jacqueline and Cory) produced an affected child (Marie).