Molecular Diagnosis of Genetic Diseases

Molecular Diagnosis of Genetic Diseases
Case 2: Sickle Cell Anemia
Sickle cell anemic R.B.Cs can be observed through simple microscopy but in case of molecular diagnosis the methods which can be used are
  1. ARMS most useful
  2. OLA
  3. Pyrosequencing (Costly)
  4. Do Blot
All these can be used to study the paint mutation. The paint mutation has to be known i.e Glutamic acid---Voline at position 6 in B-chain.

RELP can also be carried if the restriction site is being removed or added. Example
Enzyme Cvn1, restriction site CCTNAGG, mutation CCTGTGG.

Hemoglobin has this target sequence of Cvn1.
In the case of sickle cell, anemia restriction site removal occurs.

Normal AA Heterozygous As (carrier) Homozygous SS
382 ● - ● -
256 ● - ● - ● -
201 ● - ● -
181 ● - ● -
88 ● - ● - ● -

B-globin gene has three restriction sites of Cvn1. In a normal case, it gives four bands.

Two chromosomes are seen at a time of two genes.

  ↓          ↓         ↓
             ↓          ↓ 

Due to the removal of one restriction site a bigger fragment forms. The intensity of bands will be different. Product size also tells whether the condition is homozygous or heterozygous.

It can also be analyzed through amniotic fluid. (In case of lethal abortion can be carried). Through parents genotyping, the genetic disease can be diagnosed with common disorders that can be analyzed.

RNA Analysis Techniques
Methods For Quantification Of RNA

1. qPCR (costly)

2. UV based spectrophotometry based Nanodrop
Operational cost is less
Quality cannot be seen reads degraded RNA as well hence quality cannot be seen reads degraded RNA as well hence quality cannot be determined

3. MCE method
Micro capillary electrophoresis
RNA integrity can be shown

4. Fluorescence method

Inductivity coupled plasma optical emission spectroscopy.
Phosphorous emissions are analyzed quantifies the amount of phosphate group in RNA amount is seen.

6. Agarose/Polyacrylamide Gel Electrophoresis
Most convenient/cheap
Size quantity (Intact or degraded), the amount can be seen.

Two bands: 18s 23s RNA. These are intact so they come in the form of bonds.
DNA bonds will be above. mRNA is mixed as heterogeneous genes are present so a smear forms not exact clear bonds.
Rotate Your Phone Screen If You Find Irregularity In Table.

Method ICP-OES Nanodrop MCE Fluorescence
● Analysis base ● Phosphorous emissions ● UV absorbance ● Agilent Nano ● Ribogreen assay (Flourescent molecule)
● Instrument cost ● 100k$ ● 9k$ ● 25000$ ● 42,000$
● Operational cost ● 5$ ● 1$ ● 26$ ● 6-25$
● User skill ● Highest ● Lowest ● Moderate ● Moderate
● Minimum RNA amount ● 1ng ● 2ng (less sensitive) ● 0.2ng ● 0.05ng
● Advantages ● Its results are reproductive.
● Intensive to RNA quality & solvent pH.
● Rapid (2min)
● Less volume (1-2uL)
● Linear (Standard curve) can be drawn.
● Low volume.
● Gives info. about the integrity of RNA.
● The amount can be taken up to programs for analysis.
● Disadvantages ● Needs large volume for analysis 60-300ul are required.
● Time-consuming.
● ● Susceptible to impurities & plt change (phenol of DNA affect readings).5ng> ● Time Consuming (1 hour).
● Impurities & plt change also affect reading.
● Now chip for each sample.
● TIme-consuming.
● Impurities are affecting.

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