Extraction of Human DNA Experiment Goals Isolation of

Extraction of Human DNA

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

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

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 • E. coli • Human 5 -10 kb 4, 640 kb 2, 900, 000 kb

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.

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.

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.

Procedure

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

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

1 - RBCs Lysis • Pipette 3 mls of whole blood in a conical centrifuge tube • Add 9 mls of 1 X erythrocyte lysing buffer (0. 155 M NH 4 Cl, 10 m. M KHCO 3, 0. 1 m. M Na 2 EDTA, p. H 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

2 - WBCs nuclei Lysis & proteins digestion • Add 1. 5 mls of SE buffer (75 m. M Na. Cl, 25 m. M Na 2 EDTA, p. H 8. 0) containing 100µg/ml of Proteinase K & 1% sodium dodecyl sulphate (SDS) to the pellet • Incubate at 37 -55 o. C overnight in a water bath or incubator • WBCs nuclei denatured & DNA goes out in solution

3 - Separate contaminants from DNA • After incubation add 1. 5 mls of SE buffer, 750 µl of 6 M Na. Cl & 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

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

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

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

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.

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

Nucleic Acid Characterization • Absorption Spectra – Absorb light in ultraviolet range, most strongly in the 254 -260 nm range • Useful for quantification of samples

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/m. L X 0. 1 m. L = 15 µg Concentration from UV Spec. (µg DNA per ml of hydrating solution) Volume of hydration solution DNA yield

Spectrophotometric analysis of DNA

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

Quality from UV Spectrophotometry A 260/A 280 = measure of purity (A 260 – A 320)/(A 280 – A 320) 1. 7 – 2. 0 = good DNA or RNA <1. 7 = too much protein or other contaminant

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