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.
Capable of self-replication and synthesis of RNA.
Contains instructions for our body cells to perform their specific functions.

4. RNA. DNA consists of two long chains of nucleotides twisted into a double helix
Joined by hydrogen bonds between the complementary bases adenine and thymine or cytosine and guanine.
The sequence of nucleotides determines individual hereditary characteristics.

5. DNA plays an important role in two processes
The process of replication, DNA provides information to copy itself, so genetic information can be passed on from generation to generation of cells.
also provides instructions for making proteins, which are vital to the maintenance and function of cells.

6. 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
(about 2.9 billion base pairs)

7. Only about 5% code for protein
Only about 5% code for protein. Intervening sequences and other noncoding sequences make up the remainder.
In addition to genomic DNA, mitochondria, which are cellular organelles, contain their own DNA (mitochondrial DNA) which replicate independently from cell chromosomal DNA.

8. 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.

9. 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.

10. 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.
Extracted DNA is soluble in water and thus will appear as a clear solution. By contrast DNA is insoluble in salt solutions and alcohol, where it will form white fibers. Purification procedures for DNA usually include precipitation with alcohol in the presence of salt.
Once extracted, DNA can be stored for long periods of time.

11. Analysis of the isolated DNA

12. 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

13. 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.
EDTA has high affinity towards divalent ions like Ca2+, Mn2+, Mg2+ which are cofactors for many active enzymes inside the cells. That includes nucleases which digests DNA molecules. Once the cell is

14. 1- RBCs Lysis
Pipette 3 mL of whole blood in a conical centrifuge tube.
Add 9 mL of 1X erythrocyte lysing buffer.
Leave 10 min. at RT, mix occasionally.
Centrifuge at 4000 rpm for 5 min.
Discard supernatant.
White pellet is observed at bottom of tube.
Wash pellet 3 times by adding 3 mL of buffer, incubate 10 min at RT, & centrifuge
lysing buffer (0.155M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4)

15. 2- WBCs nuclei Lysis & proteins digestion
Add 1.5 mL of SE buffer to the pellet
Incubate at 37-55oC overnight in a water bath or incubator
WBCs nuclei denatured & DNA goes out in solution
SE buffer (75mM NaCl, 25 mM Na2EDTA, pH 8.0) containing 100µg/ml of Proteinase K & 1% sodium dodecyl sulphate (SDS)
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.
a protease (proteinase K) to digest the proteins

16. 3- Separate contaminants from DNA
After incubation add 1.5 mL of SE buffer, 750 µl of 6M NaCL & 3.75 mL 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

17. 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 micro centrifuge tube containing 1 mL of 75% ethanol.
Wash by inversion to remove any remaining salts.
Centrifuge at g for 4 minutes and then discard supernatant.
Repeat the washing step, then centrifuge.
Remove supernatant, and dry the pellet. .

18. 5- Resuspend DNA in final buffer
Dried pellet is resuspended in TE buffer and left overnight on a rotator
DNA Analysis
Different methods for assessing quantity & quality of extracted DNA
Agarose gel electrophoresis
UV spectrophotometry
TE buffer (1M Tris-HCl; 0.5M EDTA; pH 8.0)

19. 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

20. 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
Electrophoreses at 70–80 volts, 45–90 minutes.
view on UV transilluminator.
A good quality DNA is represented by a sharp band near the wells of the gel, while smearing indicates DNA degradation.

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

22. Nucleic Acid Characterization
Absorption Spectra
Absorb light in ultraviolet range, most strongly in the nm range
Useful for quantification of samples
Quantity: Nucleic acids have a peak absorbance in the ultraviolet range at about 260 nm

23. 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

24. Spectrophotometric analysis of DNA

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

26. 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

27. 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
TE buffer "TE" is derived from its components: Tris, a common pH buffer, and EDTA,