Immuno Quantitative Assay

Immuno quantitative Assay

• 1000 fold more sensitive than EUSA

DNA will be made to bind with biotin-labeled probes that are bound to Streptavidin. Then the DNA probe of size 246bp will bind to it. Unbound probes are then washed outbound probes will be amplified with the help of PCR. Signal increases to a million times. It is then detected with syber green or ethidium bromide. A thing that couldn’t be tested with ELISA was amplified 10,000 times could be detected. The double strand-specific dye will be used. It is a good technique to increase sensitivity levels.

Animal Species Determination

The enzyme in mitochondrial DNA ‘cytochrome b’. This gene (Just like ribotyping is specie specific) is specie specific being mitochondrial DNA. Mitochondrial DNA will have more copies; even if the sample size is less it can be analyzed (Genomic DNA copies will be less).

• To sop trafficking of endangered animals.

• To stop illegal use of hydes, teeth, bones

480bp sequence of cytochrome b is specie specific. The blast can be used. Bones; hairs (fallide cells) have enough amount of mitochondrial DNA for detection.

Molecular Diagnosis of Genetic Diseases

Case 1: Cystic Fibrosis

• Common in the European/White population.

• 1400 mutations reported.

• 🔺F508

Such mutations will be important which are responsible for the change in the amino acid at different positions. There are different mutations but 90% are deletion at position 508 i.e phenylalanine is not present. There are 98% chances of figuring which mutation is present. 20 are the most common.

CFTR ➡️ Cystic Fibrosis Transmembrane Conductance Regulator protein is involved in the movement of Na+ and Cl- ions; maintaining gradient. Most amino acids helping in its structure and function are mutated.

Mutation does not occur again and again; deletion occurred only once. It is being transferred. Carriers will be more. Recessive disorder, not dominant. If homozygous then disease. More mucous produces, bacteria entangle more, infection occurs more, respiratory problems.

• Its mutation is transferred so 90% have the problem.

• 100 people ➡️ all cystic fibrosis ➡️ 100% hereditary ➡️ 1 frequency.

• 50% have, 50% not ➡️ 0.5% frequency.

• If 1% of individuals have this then affected individuals will be less. It also depends on gene frequency.

e.g:
Sickle cell anemia was helping in malarial protection. All those individuals who were normal died of malaria. In this way the frequency of sickle cell population increase as this gene made them fit to survive in all population. Thus population dynamics change.

Different methods can be used for detection.

1. Dot Blot Method: 
DNA is amplified, the CFIR gene will be amplified. It is then transferred to the membrane and is hybridized to a labeled oligonucleotide probe. Conditions should be stringent here. Even if there is a difference of 2-3 nt then probe should not bind e.g in case of phenylalanine as one codon is less so there should be such probe which binds with normal but not mutant. In this way, a mutation can be detected.
 
2. ARMS: 
As all mutations are not important (21 are important out of these further 1 is more important) so it can be seen that which person is Camier. Through genetic counseling it can be observed that which kid will have this

Camer =cC       cc=diseased     CC=normal 

• If one individual is heterozygous normal i.e carrier and other is diseased then 50% kid will have it and 50% will be carriers.

• If an individual is normal and the other is diseased then all will be carriers.

• If both individuals are carriers then 50% will be carriers, 25% diseased, and 25% normal.

• If both are diseased then there are 100% chances of diseased i.e all kids will have it.

3. PCR/OLA: 
Technique is like ARMS but involves ligation as well. (Ohganucleotidel ligation assay)
3' end nucleotide matches with primer ➡️ product. Provided that primers are designed for the known position.

Ligose will not add any nucleotide. It will seal the nick by creating a phosphodiester band between. Then this part will be bound to streptavidin.

This method can be used in both senses: 

1. The desired mutation is present.
2.  Some/any mutation is present.

PCR-OLA is most useful for the detection of mutation especially in case of diagnosis of genetic diseases.

Editor's Recommendation:

• Analysing Metabolic Pathways
• Protein Threading Sequence
• Ab Initio Protein Structure Prediction
• Homology Modeling
• Hot Start PCR, Multiplex PCR, Avoiding Contamination In PCR, Advantages, and Disadvantages in PCR
• DNA Damage
• Docking | Protein-Protein Docking | Protein-Ligand Docking 
• Functional Regulation | Genetic Aspect | Indirect Aspects 
• Database Development 
• Functional Analysis At Structure Level
• PTMs and Functional Regulations
• Modeling Cellular Processes
• PCR Reagents | Stochastic Effect | STR Classification
• DNA Degradation
• DNA Quantification | Human DNA Quantification Method | Advantages  
• Desirable Characteristics of STR used in Forensic DNA typing
• DNA Ladders
• Metabolic Pathways
• Non-Human DNA
• Mitochondrial DNA 
• Integrated Genomic Circuits 
• Shutter Product Formation 
• Mini STR Sites 
• Real-Time PCR
• Molecular Diagnosis of Genetic Diseases
• STR Sites