NUCLEIC ACID DNA AND RNA ISOLATION MOLECULAR BIO.

Cabucana, Paul lester T. LAB

MOLECULAR DIAGNOSTICS
NUCLEIC ACID ISOLATION
Specimen Collection  
a. Whole blood.  
- This is the most common type of molecular diagnostic specimen. 
- Blood is most commonly collected in purple-top tubes.  
- Heparin is a potent inhibitor of PCR and is, therefore, not used in molecular diagnostics.  
- Blood should be stored at 4◦C until processing.  
 
b. Tissue. 
- Tissue specimens should be frozen immediately upon receipt or held in cell culture medium at 37◦C until processing.  
 
c. Buccal swabs. 
- Often used for remote site testing; collection of DNA specimens for parentage testing.  
- Swabs should be air-dried before shipment to the laboratory.  
 
d. Other specimens include those also commonly encountered in the clinical laboratory including urine, feces, sputum, cerebrospinal
fluid, genital swabs, semen, etc. Other molecular diagnostic specimens include sections of formalin-fixed paraffin, embedded tissues,
bone, hair, and fingernails (usually confined to forensic specimens). 

DNA ISOLATION
Phenol:Chloroform:Isoamyl alcohol method.  
- It is the “gold standard.” Yields a large mass of high-quality DNA. Can be used to isolate DNA from any specimen type. This method
normally requires a large mass of starting material.  
 
Manufacturer (Qiagen) kits.  
- Provide ease of use and high degree of reproducibility. Yields smaller amounts of DNA. DNA is usually very pure. Most often used to
isolate DNA from blood. This is the most commonly encountered clinical DNA isolation method.  
 
Quick-extract solutions.  
Fastest methods. Low quality, low mass DNA recovery. Usually used to isolate DNA from buccal swabs/FTA cards. 

BASIC STEPS IN SOLATING DNA FROM CLINICAL SPECIMENS 

1. Separate WBCs from RBCs, if necessary 
2. Lyse WBCs or other nucleated cells 
3. Denature or digest proteins 
4. Separate contaminants (e.g., proteins, heme) from DNA 
5. Precipitate DNA, if necessary 
6. Resuspend DNA in final buffer 
METHODS:
1. Liquid phase organic extraction (phenol/chloroform)
2. Liquid phase non-organic extraction
3. Solid phase procedures
RNA ISOLATION
a. Acid phenol/LiCl salt isolations.
- Gold standard. Yields a large mass of high- quality RNA. Can be used on any specimen type.
b. Manufacturer kits.
- Provide ease of use and high degree of reproducibility. Preferred method for clinical RNA isolation.
c. Poly-A enrichment kits.
- Increase recovery of mRNA in specimens.
 DNA or RNA quantity, quality and molecular size are characterized by:
a. Spectrophotometry
b. Agarose gel electrophoresis
c. Fluorometry
d. Colorimetric blotting

MOL. BIO NUCLEIC ACID, DNA and RNA Isolation Page 1 of 2

 NUCLEIC ACID DNA AND RNA ISOLATION MOLECULAR BIO.
Cabucana, Paul lester T. LAB

GEL ELECTROPHORESIS
  method used to separate DNA fragments onto a solid matrix based upon size.   
1. Agarose gel electrophoresis is most often performed to resolve DNA fragments larger than 100 bp in size.  
2. Polyacrylamide gel electrophoresis is the method of choice to resolve small DNA fragments. Very high concentrations of
polyacrylamide matrix can resolve down to 1 bp differences in size. 
3. Capillary gel electrophoresis is a modification of polyacrylamide gels used in automated DNA sequencing and genotyping
platforms. 

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