Biochemistry Flashcards
Properties of vectors (2C)
Capable of replication inside the host cell
Contains at least 1 restriction site for 1 restriction endonuclease.
Types of vectors
Plasmids: Double stranded circular/small. Clone small DNA fragments up to 10 kb.
Phage: Virus, clones large DNA fragments 20 kb
Cosmid: responsible for packing DNA, up to 50 kb
Process of cloning consists of
Plasmids from bacteria/ foreign DNA are cut by restriction endonuclease
The cohesive ends of foreign DNA recombine with plasmid and joined by ligase to form recombinant DNA
Recombinant DNA is introduced into bacterial host cell. Uptake of DNA is called transformation
The transformed bacterial cell grow and divide and so does the recombinant plasma DNA
Requirements for PCR: TD TD B
- Two DNA primers.
- Deoxyribonucleoside triphosphate (dATP)
- Thermostable DNA polymerase (taq polymerase).
- DNA to be amplified.
- Buffer solution.
Polymerase chain reaction (PCR)
In vitro method for DNA amplification
PCR steps
a. Denaturation: Heating to 95° C.
b. Primer annealing: Cooling to 50° C.
c. Elongation: Heating 72 C allowing taq to elongate the primers. After 10 cycles DNA amplified 1000 times, after 30 cycles DNA amplified 10^9
Apparatus in PCR heating cooling heating is called
automated thermocycler
Advantages of PCR
Sensitive
Faster
Technically less difficult
Applications of PCR: 2S 2P FG
- Synthesis of DNA for sequencing and cloning.
- Synthesis of proteins: insulin - GH -Vaccines - monoclonal Ab.
- Diagnosis of genetic diseases as sickle cell anemia.
- Detection of bacterial and viral infection i.e., HIV and hepatitis B.
- Forensic medicine: amplification of DNA from hair or sperm.
- Gene therapy.
Hemoglobinopathies from Production of abnormal hemoglobin:
* Sickle cell anemia (HbS disease).
* HbC disease.
* HbM disease (Methemoglobinemia).
Hemoglobinopathies from Decreased Production of normal hemoglobin:
* alpha Thalassemia: Defect in production of the alpha chain.
* Beta Thalassemia: Defect in production of the beta chain.
Sickle cell anemia (HbS disease)
a type of Point mutation;
There are 2 genes for the B chain so, Patient may be:
* Homozygous for sickle cell anemia: Contains Hb S only.
* Heterozygous for sickle cell anemia (sickle cell trait): Contains Hb A and Hb S.
Thalassemia
Hereditary hemolytic diseases due to gene mutation or deletions.
alpha Thalassemia:
There are 4 a genes (2 on each chromosome 16).
a. Defect in 1 gene -> carrier for a thalassemia.
Symptoms: Completely normal (silent carrier).
b. Defect in 2 genes -> a thalassemia trait.
Symptoms: Mild anemia.
c. Defect in 3 genes -> a thalassemia major.
Symptoms: Severe anemia.
d. Defect in 4 genes -> Homozygous for a thalassemia.
Effects: - Dies intrauterine (Hydrops fetalis).
Beta thalassemia
There are 2 beta globin genes
Defect in 1 gene –> Beta thalassemia minor (trait)
Symptoms: mild anemia
Defect in 2 gene—–> Beta thalassemia major
Symptoms: severe anemia later on after birth
Patient with p thalassemia major appears normal at birth …. Explain why?
Due to presence of HbF
High levels of HbF and HbA2 in patient with p thalassemia………….Explain why?
To compensate the absence of HbA.
Respiratory (Volatile) acids
as carbonic acid H2CO3, excreted by lungs
Metabolic (Fixed) acids:
Not excreted by the lungs as:
Sulfuric acid/ phosphoric acid
Uric acid and nucleic acid
lactic acid
Mechanisms of regulation of pH:
- Buffers:
- 1st line of defense
- Acts in a fraction of a second (immediate response) - Respiration (Ventilation):
- By increase/decrease CO2 excretion so regulates the amount of H2CO3 in the body - Kidneys:
- 2nd line of defense
- Slow process (takes several days to reach maximum capacity)
- By excretion of excess acids (H+) or bases (HCO3)
Buffers can be
A weak acid and its salt with strong base
H2CO3/ Na bicarbonate (NAHCO3)
Acetic acid/ Na acetate (CH3COOH/CH3COO)
A weak base and its salt with strong acid
Ammonium hydroxide/ ammonium chloride
Physiological Buffers types
- Bicarbonate buffer system: mainly in Extracellular fluid
- Phosphate buffer system: in all types of cells
- Protein buffer system: in cells and plasma
- Hemoglobin buffer system: in RBCs and Specific for buffering CO2 Produced by oxidation in tissues
Bicarbonate buffer system:
BHCO3/H2CO3, ratio 20:1, main buffer system in lungs
Advantages of Bicarbonate buffer system:
- Present in higher concentrations than other buffers
- Easily formed at the tissues from CO2 by Carbonic Anhydrase enzyme
- Easily corrected by respiration; CO2 is excreted with expired air at Lungs