Isolation of Genomic DNA

S. KARUTHA PANDIAN
DEPARTMENT OF BIOTECHNOLOGY
(DST-FIST Sponsored Department)
ALAGAPPA UNIVERSITY
(A State University Accredited with ‘A’ Grade by NAAC)
KARAIKUDI-630 003
Nucleic Acid Preparation
Sample Source?
 Amniocytes or
Bacterial Culture amniotic fluid
Whole blood
 Dried blood spots
 Buccal cells
 Fresh or frozen tissue
 Cultured cells
(biopsy material)
 Sputum, urine, CSF, or
other body fluids
 Fixed or paraffin-
embedded tissue
Nucleic Acid Preparation
Choosing an Isolation Method
 Important factors are:
 Processing speed
 Ease of use
 Yield of DNA or RNA
 Quality of DNA and RNA prepared (amplification
performance)
 Shelf life/storage conditions
 Quality assurance criteria
 Cost of preparation
Basic Steps in Isolating DNA
Harvesting of cells
Lyse cells
Denature/digest proteins
Separate contaminants (e.g., proteins, heme)
from DNA
Precipitate DNA if necessary
Resuspend DNA in final buffer
DNA Isolation Methods
Liquid Phase Organic Extraction
 Phenol (50):chloroform/isoamyl alcohol (50:49:1)
 Lysed samples mixed with above; two layers are formed.
 Proteins remain at interface.
 DNA is removed with top aqueous layer.
 DNA is precipitated with alcohol and rehydrated.
 Disadvantages:
 Slow, labor-intensive, toxic (phenol, chloroform)
 Fume hood required, disposal of hazardous materials
required
DNA Isolation Methods: Liquid Phase
Nonorganic Salt Precipitation
 Cell membranes are lysed and proteins are denatured
by detergent (such as SDS).
 RNA is removed with RNase.
 Proteins are precipitated with salt solution.
 DNA is precipitated with alcohol and rehydrated.
 Advantages:
 Fast and easy method
 Uses nontoxic materials, no fume hood required, no
hazardous materials disposal issues
 Produces high-quality DNA
Resuspending Final Nucleic Acid
Samples
 Have some idea of expected nucleic acid yield.
 Choose diluent volume according to desired
concentration.
 Calculating Expected DNA Yield
 Example:
1 X 107 cells X 6 pg DNA/cell X 80% yield= 48 ug DNA
 Resuspend DNA in TE buffer or ultra pure DNAse-
free water.
 Resuspend RNA in ultra pure RNase-free water.
Nucleic Acid Analysis
 DNA or RNA is characterized using several
different methods for assessing quantity,
quality, and molecular size.
 UV spectrophotometry
 Agarose gel electrophoresis
 Fluorometry
 Colorimetric blotting
Quantity from UV Spectrophotometry
 DNA and RNA absorb maximally at 260 nm.
 Proteins absorb at 280 nm.
Quantity from UV Spectrophotometry
 [DNA] =
A260 X dilution factor X 50 µg/mL

 [RNA] =
A260 X dilution factor X 40 µg/mL
 Concentration = µg of DNA or RNA per mL
of hydrating solution
Quantity from UV Spectrophotometry
Calculating Yield
Multiply the concentration of the
DNA or RNA sample by the
volume of hydrating solution added.
Example for DNA: 150 µg/mL X 0.1 mL = 15 µg

Concentration from UV Volume of DNA yield

Spec. (µg DNA per ml hydration
of hydrating solution) solution
Quality from UV Spectrophotometry
A260/A280 = measure of purity

1.7 – 2.0 = good DNA or RNA

<1.7 = too much protein or
other contaminant (?)
Quality from Agarose Gel
Electrophoresis
 Genomic DNA:
 0.6% to 1% gel, 0.5µg/mL ethidium bromide in
gel and/or in running buffer
 Electrophorese at 50 volts, 45–90 minutes.
DNA Size from Agarose Gel Electrophoresis:
Compares unknown DNA to known size standards

Lambda DNA cut 1 kb ladder

Lambda DNA with Hind III 12,218 bp
100 bp ladder
6,018 bp
23,130 bp
1,500 bp
9,416 bp 3,054 bp 1,000 bp
6,557 bp 2,036 bp 600 bp

4,361 bp 1,636 bp
300 bp
48,500 bp 1,018 bp
(48.5 kb)
100 bp
2,322 bp
2,027 bp 517 bp
DNA Quality from
Agarose Gel Electrophoresis
 High molecular weight band (>48.5 kb)
 Smearing indicates DNA degradation (or too
much DNA loaded).
DNA Quality from Agarose Gel
Electrophoresis
Human Whole Blood DNA

Lambda DNA
marker Genomic DNA
Lambda DNA cut with
Hind III marker
Storage Conditions
 Store DNA in TE buffer at 4 °C for weeks or
at –20 °C to –80 °C for long term.
Troubleshooting Nucleic Acid
Preparation Methods
 Problem: No or low nucleic acid yield.
 Make sure that ample time was allowed for
resuspension or rehydration of sample.
 Repeat isolation from any remaining original
sample (adjust procedure for possible low cell
number or poorly handled starting material).
 Concentrate dilute nucleic acid using ethanol
precipitation.
Troubleshooting Nucleic Acid
Preparation Methods
 Problem: Poor nucleic acid quality
 If sample is degraded, repeat isolation from
remaining original sample, if possible.
 If sample is contaminated with proteins or other
substances, clean it up by re-isolating
(improvement depends on the extraction
procedure used).