Presentation on theme: " 1. Blood Groups: Each of the blood group system is determined by a different gene or set of genes. The various antigens that can be expressed within."— Presentation transcript:
1. Blood Groups: Each of the blood group system is determined by a different gene or set of genes. The various antigens that can be expressed within a system are the result of different DNA sequences in these genes. Two blood systems that have special medical significance, the ABO and Rh systems. The ABO system consists of two major antigens, labeled A and B located on the surface of erythrocytes. Individuals can have one of four blood types depends on presence or absence of one or both antigens.
The ABO system is encoded by a single gene on chromosome 9, consists of three primary alleles, labeled I A, I B, and I O. Individuals with I A allele have the A antigen on their erythrocyte surface (blood type A), Those with I B have the B antigen on their cell surface (blood type B). Those with both alleles express both antigens (blood type AB), and those with two copies of I O allele have neither antigen (type O blood), because the I O allele produces no antigens.
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1. ABO blood system used in studies of genetic variation among individuals and populations. 2. Determining the compatibility of blood transfusions and tissue grafts. 3. Some combinations of these systems can produce maternal- fetal incompatibility with serous results for the fetus (Rh- system)
The principle of this technique is to detect variations in DNA, RNA, and variations in serum proteins that encoded by certain genes. These variations are observable because all (DNA, RNA, Protein) can be separated by means of an electric field. Clinical Application: To determine whether an individual has normal hemoglobin (HbA) or the mutation that causes Sickle cell disease (HbS). The replacement of glutamic acid with valine in the ß- globin chain produces a difference in electrical charge.
The hemoglobin is placed in an electrically charged gel composed of starch or agarose. The slight difference in charge resulting from amino acid replacement causes the HbA and HbS forms to migrate at different rates through the gel. After several hours of migration, the protein then stained with chemical solutions so that their positions can be seen. So polymorphism can detected if the HbA is homozygote or HbSS homozygote, or having a heterozygote HbA on one chromosome and HbS on the other.
The rate of variation in human DNA occurs at an average of 1 in every 300 to 500 base pair (bp). Thus, approximately 10 million polymorphisms may exist among the 3 billion bp that comprise the human genome. Fortunately, molecular techniques developed during the past 30 years enable the detection of thousands of new polymorphisms at the DNA level. These techniques includes:
Principle: PCR making millions of copies of a short, specific DNA sequence very quickly. Heating- cooling cycles are used to denature DNA and then build new copies of a specific, primer- bounded sequence. Clinical Application: Because of its speed and ease of use, this technique is now widely used for: Assessing genetic variation for diagnosis genetic diseases forensic purpose detection and diagnosis of infectious diseases used as fingerprints to identify genetic relationship between individuals, such as parent- child or between siblings, and are used in paternity testing
PCR instruments includes: Thermocycler PCR Agarose gel electrophoresis PCR process requires four components: 1. Two primers: each consisting of 15-20 bases of DNA, containing sequences complementary to the 3’ end of target region of DNA that contains the polymorphism or a mutation that causes disease. 2. Heat- stable DNA polymerase enzyme: originally isolated from the bacterium Thermus aquaticus with a temperature optimum at round 70 C. 3. A large number of free DNA nucleotides (dNTPs). 4. Small quantity of Genomic DNA from an individual act as a template.
Typically, PCR consists of a series of 20- 40 repeated temperature changes called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps usually three. 1. Denaturation step: The genomic DNA is first heated to a temperature of 94-98 C for 20-30 seconds. It causes melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single- stranded DNA molecules.
2. Annealing step: The reaction temperature is lowered to 50-65 C for 20-40 seconds allowing annealing of the primers to the single – stranded DNA template. Stable DNA-DNA hydrogen bonds are formed when primer sequence closely matches the template sequence. The polymerase enzyme binds to the primer- template hybrid and begins DNA synthesis
3. Extension/ elongation step: Taq polymerase has its optimum activity temperature at 75- 80 C, and commonly a temperature of 72 C is used with this enzyme. At this step the DNA polymerase synthsizes a new strand complementary to the DNA template strand by adding dNTPs in 5’ to 3’ direction. The DNA polymerase will polymerize a thousand bases per minute.
Finally agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker) which contains fragments of known size, run on a gel alongside the PCR products.
Is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites. It took advantage of the existence of bacterial enzymes known as restriction endonucleases or restriction enzymes. These enzymes are produced by various bacterial species to “restrict” the entry of foreign DNA into the bacterium by cutting or cleaving the DNA at specifically recognized sequences. These sequences are called restriction sites. For example, Escherichia coli produces a restriction enzyme called EcoR1, that recognizes the DNA sequence GAATTC so this enzyme cleaves the sequence between the G and the A, this produces DNA restriction fragments.
The RFLP process: First DNA is extracted from blood samples then digested by a restriction enzyme and then loaded on a gel. Electrophoresis separate the DNA fragments according to their size. The DNA is denaturated and transferred to a solid membrane and hybridized with a radioactive probe. Exposure to x-ray film appears specific DNA fragments (bands) of different sizes in individuals.
The cloning of a gene produce many identical copies. Recombinant DNA technology is used when a very large quantity of the gene is required. Recombinant DNA (rDNA) contains DNA from two or more different sources. It required a vector to introduce the rDNA into a host cell. One common type of vector is plasmid. Plasmids are small accessory rings of DNA from bacteria. The ring is not part of the bacterial chromosome and replicates on its own.
Two enzymes are needed to introduce foreign DNA into vector DNA. The first enzyme, called a restriction enzyme, cleaves the vector’s DNA, and the second, called DNA ligase, seals foreign DNA into the opening created by the restriction enzyme. The single-stranded, but complementary, ends of the two DNA molecules are called “sticky ends” because they can bind a piece of foreign DNA by complementary base pairing
Bacterial cells take up recombinant plasmids. Thereafter, if the inserted foreign gene is replicated and actively expressed, the investigator can recover either the cloned gene or a protein product. Example: Cloning of the human Insulin gene in bacterial cell.