1. Extraction of Human DNA

2. Experiment Goals Isolation of genomic DNA from human blood
Analysis of isolated DNA using
Agarose gel electrophoresis
Spectrophotometry

3. What is a DNA? DNA, also known as deoxyribonucleic acid,
A fundamental molecule found in all living things
Carries the genetic information in the cell
Contains instructions for our body cells to perform their specific functions
The sequence of nucleotides determines individual hereditary characteristics

4. What is a DNA? Basic unit of information in DNA is the gene
Human beings have about 30,000 gene
Size of organism’s genome is roughly a measure of its complexity
Viruses 5-10 kb
E. coli 4,640 kb
Human 2,900,000 kb

5. DNA Extraction
DNA extraction is a routine procedure to isolate & collect DNA.
DNA extraction is the first step for subsequent molecular or forensic analysis.
DNA can be extracted from almost any intact cellular tissue
Skin,
blood,
saliva,
semen,
mucus,
muscle tissue,
bone marrow, etc.

6. Nucleic Acid Preparation Applications
Medical studies
Understanding genetic disorders at molecular level
Rapid detection of genetic disorders in a patient
Agricultural studies
Plant and animal breeding
Criminology/Paternity testing
DNA fingerprinting to identify individuals.

7. Basic steps in DNA extraction
There are three basic steps in a DNA extraction, the details of which may vary depending on the type of sample and any substances that may interfere with the extraction and subsequent analysis.
Break open cells and remove membrane lipids
Remove cellular and histone proteins bound to the DNA, by adding a protease, by precipitation with sodium or ammonium acetate, or by using a phenol/chloroform extraction step.
Precipitate DNA in cold ethanol or isopropanol, DNA is insoluble in alcohol and clings together, this step also removes salts.

8. Procedure

9. Overview of Procedure 1- Lyse RBCs & WBCs
2- Lyse WBCs nuclei & Denature/digest proteins
3- Separate contaminants (e.g., proteins, heme)
4- Precipitate DNA
5- Resuspend DNA in final buffer

10. Blood Collection Blood collected in disodium EDTA tube
Samples can be stored at -20oC or -70oC
Fresh samples are kept in freezer for a few hours to facilitate RBCs hemolysis
Allow samples to thaw before starting the extraction

11. 1- RBCs Lysis
Pipette 3 mls of whole blood in a conical centrifuge tube
Add 9 mls of 1X erythrocyte lysing buffer (0.155M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4)
Leave 10 min. at RT, mix occasionally
Centrifuge at 4000 rpm for 5 min
Discard supernatent
White pellet is observed at bottom of tube
Wash pellet 3 times by adding 3 mls of buffer, incubate 10 min at RT, & centrifuge

12. 2- WBCs nuclei Lysis & proteins digestion
Add 1.5 mls of SE buffer (75mM NaCl, 25 mM Na2EDTA, pH 8.0) containing 100µg/ml of Proteinase K & 1% sodium dodecyl sulphate (SDS) to the pellet
Incubate at 37-55oC overnight in a water bath or incubator
WBCs nuclei denatured & DNA goes out in solution
NaCl so that DNA is stabilised and remains as a double helix,
EDTA which chelates Mg++ and is a co-factor of DNase which destroy up DNA rapidly,
anionic detergent SDS which disrupts the lipid layers, helps to dissolve membranes & binds positive charges of chromosomal proteins (histones) to release the DNA into the solution.
Include a protease (proteinase K) to digest the proteins

13. 3- Separate contaminants from DNA
After incubation add 1.5 mls of SE buffer, 750 µl of 6M NaCl & 3.75 mls chloroform
Mix vigorously on vortex for 20 sec
Mix for 30 min (on rotator)
Centrifuge for 10 min at 2000 rpm
2 phases are observed
DNA is extracted in supernatant & proteins in the lower phase
Transfer upper aqueous phase (containing DNA) to a clean tube
Organic solvent extraction- chloroform
addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA

14. 4- Precipitate DNA Add an equal volume of isopropanol
DNA will be precipitated by gentle swirling & observed as a white thread like strand
Using a sterile spatula or loop transfer the DNA strand into a sterile microcentrifuge tube containing 1 ml of 75% ethanol
Wash by inversion to remove any remaining salts
Centrifuge, discard supernatent
Repeat the washing step, then centrifuge
Remove supernatant, and dry the pellet .

15. 5- Resuspend DNA in final buffer
Dried pellet is resuspended in TE buffer and left overnight on a rotator

16. DNA Analysis
Different methods for assessing quantity & quality of extracted DNA
Agarose gel electrophoresis
UV spectrophotometry

17. Checking the Quality of DNA
The product of DNA extracted will be used in subsequent experiments
Poor quality DNA will not perform well in PCR

18. Quality from Agarose Gel Electrophoresis
Quality of DNA extracted is assessed using the following simple protocol:
Mix 5 µL of DNA with 5 µL of loading Dye
Load this mixture into a 1% agarose gel
Stain with ethidium bromide
Electrophorese at 70–80 volts, 45–90 minutes.

19. DNA Quality from Agarose Gel Electrophoresis
High molecular weight band
Smearing indicates DNA degradation

20. Nucleic Acid Characterization
Absorption Spectra
Absorb light in ultraviolet range, most strongly in the nm range
Useful for quantification of samples

21. Quantity from UV Spectrophotometry Calculating Yield
Multiply the concentration of the
DNA sample by the
volume of hydrating solution added.
Example for DNA: 150 µg/mL X 0.1 mL = 15 µg
Concentration from UV Spec. (µg DNA per ml of hydrating solution)
Volume of hydration solution
DNA yield

22. Spectrophotometric analysis of DNA

23. Quality from UV Spectrophotometry
DNA absorb maximally at 260 nm.
Proteins absorb at 280 nm.
Background scatter absorbs at 320 nm.

24. Quality from UV Spectrophotometry
A260/A280 = measure of purity
(A260 – A320)/(A280 – A320)
1.7 – 2.0 = good DNA or RNA
<1.7 = too much protein or
other contaminant

25. Storage Conditions
Store DNA in TE buffer at 4 °C for weeks or at –20 °C to –80 °C for long term.
2–25 °C
2–8 °C
–20 °C
–70 °C
Recommended
for long-term
storage in ethanol
<4 Months
1–3 Years
<7 Years
>7 Years