Block 2 Dr. Burns

Question 1

Sickle Cell Disease (Case II)

Answer 1

• Low Hemoglobin
• High Reticulocytes
• Hemolytic Anemia
• Blood Film (a) Show sickled erythrocytes.
• Bony infarctions can cause unequal finger length

Prognosis:

• High Likelihood of crises necessitating hospital admission, transfussion, prophylactic penicillin

Facts:

• 1 in 365 African americans
• 1 in 16, 300 Hispanic Americans
• Autosmal Recessive
• Caused by a A -> T transversion in the HBB encoding B-Globin
• Amino Acid 6: Glutamic Acid (E) -> Valine (V) . E6V
  
  • Results in abnormal hemoglobin (HbS)
    
    • Normal adult Hemoglobin in HbA

Clinical Presentation:

• Presents within first 2 years
• Severe Hemolysis
• Crises lastin 5 to 7 days
• Hemolytic Anemia, failure to thrive, ,splenomegaly
• Painful swelling of hands/feet ( occlusion of Capillaries.
• Repeated Infections
  
  • Infection is a major cause of Death.

Treatments:

• Hydroxyurea
• Supporting including O₂ Transfussion, Penicillin

Question 2

Co-Occurrence of the Sickle Cell Allele and Malaria

Answer 2

The Sickle Cell allele provides protection to newborn babies and young todlers agains malaria. Approximatelly 90 % of newborns die of maleria, but the sickle cell allele protect them from the parasite malaria.

Question 3

Sickle Cell Trait

Answer 3

• Heterozygotes (ie. Carries)
• Trait: generally healthy but may be symptomatic under stressful conditions
• 8- 10% African Americans
• ~ 25% in West Africa.
• Individuals do generally have a sub-clinical anemia that can be detected in a complete blood count (CBC)

Question 4

Restriction Endonucleases.

DNA Nucleases

Answer 4

• Digest DNA
  
  • Cleave Phosphodiester bondes between nucleoties.
• Exonucleases digest from an end
• Endonucleases digest at an internal site.

Question 5

Restriction Endonucleases

Restrictions Enzymes

Answer 5

• Recognize a specific DNA sequence and cleave a phosphodiester bond in both strands within that sequence.
• Highly Specific.
  
  • Always cleaves at the same Short DNA sequence.
  • Only cleaves within that 4-8 bp sequence.
• Biological Role in bacterias as a component of the restriction/ Modification system.
  
  • A Bacterial defence mechanism against bacteriophage

Question 6

Restriction Endonucleases

Sticky Ends/Cohesive Endes.

Answer 6

• The restriction enzyme produces a staggered cut generating a 3’ or 5’ single-stranded overhang
• The overhangs can anneal with the complimentary overhang from another end (They are Sticky)

Blunt Ends:

• The restriction enzyme cuts both strands at the same position so no overhang is generated.

Question 7

Restriction Fragments.

Electrophoresis

Answer 7

1. Digestion of a DNA sample with a restriction enyme
2. Separation by gel electrophoresis
3. Staining of DNA with a RED fluorescent dye.

Question 8

The Sickle Cell Mutation Alters……

Answer 8

A restriction Site

• Wild-type allele (B^A ) contains an MstII recognition frequence
• Mutant allele (B^S) does’nt
• So we can use MstII to distinguish the alleles
• This could be the basis for a genetic test to genotype individuals and make a diagnosis.

Question 9

Hybridization Assays

Answer 9

A Nucleic acid probe of know sequence is used to detect the specific complementary sequence within a complex sample of target nucleic acid.

Question 10

Hybridization Stringency

Answer 10

• Stringency: the degree to which non complementary sequences are tolerated during hybridization.
  
  • The higher the stringency the fewer mismatches will be tolerated.
  • Some applications require high stringency.
    
    • E.g. Detecting a point mutation, a “ perfect match” is necessary.
  • Others require a low stringency
    
    • E.g. Identifying multiple genes in a gene family, “ mismatches” are tolerated.
• Stringency is determined by hybridization conditions:
  
  • Temperature
  • Salt concentration
  • Denaturing Agents (Formamide)

Question 11

Southern Blootting

Answer 11

• DNA target separated by gel electrophoresis
• Transferred onto membrane
• Detection of the DNA target using complementary DNA probe

Question 12

Northern Blotting

Answer 12

• RNA target separated by gel electrophoresis
• Transferred onto membrane
• Detection of the RNA target using complementary DNA probe.

Question 13

Western Blotting;

Answer 13

• Protein target separated by gel electrophoresis
• Transferred onto membrane
• Detection of the protein target using antibodies agains protein.