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

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.

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.

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)

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.

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. 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.

Analysis of the isolated DNA

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 -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

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)

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

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

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 11000 g for 4 minutes and then discard supernatant. 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 TE buffer (1M Tris-HCl; 0.5M EDTA; pH 8.0)

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 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.

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: Nucleic acids have a peak absorbance in the ultraviolet range at about 260 nm

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

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 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

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,

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