Laboratory Activity Nine DNA Extraction & Analysis

Purpose of Lab 09 Common Handling & Analyses: Educational – provide experience in the handling and analysis of DNA. Common Handling & Analyses: Extraction Purification Quantification Restriction digest Amplification Electrophoresis Base sequencing Cloning

DNA Extraction Cell / tissue disruption to release DNA into extraction buffer. Solubilize and remove membranes & lipids with detergents. Remove protein contamination by protease treatment or phenol extraction. Precipitate DNA with alcohol. Nucleolus Heterochromatin Euchromatin An Interphase Nucleus  30 nm  (Nucleosomes) 10 nm Phenol/chloroform extracts proteins into lower organic phase, some hang up at the interface. Nucleosome has about two loops of DNA around histone core, and approx. 150 – 200 bp’s long. Extraction buffer pH is 9.5 – alkaline to strip H+ from basic histone proteins (and AA’s – lysine & arginine), thus weakening the interaction between histone & DNA (which is negative).

Ethanol Precipitation Slowly add ice-cold ethanol (so that it “floats” on top of the DNA extract). Ethanol dilutes water, removing hydration shells of + ions, allowing DNA phosphate salts to form. DNA salts are less soluble in alcohol solutions than aqueous solutions. DNA precipitates at the water- ethanol interface. Banana Onion

Quantification of Nucleic Acids Spectrophotometric, based on absorbance at 260 nm. OD260 of 1.0 = 50 µg/mL double-stranded DNA*. 37 µg/mL single-stranded DNA. 40 µg/mL RNA. OD260 can be problematic: Protein absorbs strongly at 280 nm. Protein is the most common contaminant in DNA preparations. Purity / contamination can be estimated via OD260:OD280 ratio. 1.7 – 2.0 is good. 1.0 is bad (mostly protein). I 300 280 260 *µg/mL = ABS260 x 50

UV-260/280 Interpretations ABS260/ABS280 % DNA % Protein 2.0 1.9 1.8 1.7 1.3 1.0 0.6 100 70 50 30 10 5 90 95

Restriction Digests Definition: The enzymatic cleavage of higher MW DNA into smaller fragments with a “restriction endonuclease”. Origin & Significance of Restriction Enzymes: Defense enzymes produced by microbes. Prevent (“restrict”) the takeover by foreign (viral) DNA. Cut DNA at very specific recognition sites & patterns. Applications in Molecular Biology: Cut large cumbersome DNA molecules into smaller easily manageable fragments. “Sticky ends” are extremely useful in cloning and recombination.

“EcoR1” (Restriction Enzyme no. 1 from E. coli) “Sticky Ends”

“EcoR1” (Restriction Enzyme no. 1 from E. coli) Recipient (host) DNA Foreign (engineered) DNA Recombinant DNA

Hind III Digest of Lambda DNA Phage Lambda  DNA COS Site Lysis genes Head Tail DNA Replication Host Integration Hind III Restriction enzyme from Haemophilus influenzae. Phage  Bacteriophage that infects E. coli. One of the most studied viruses. Contains “ DNA”. Lambda DNA 48,502 bp long; circular or linear. Complete sequence known. Has 7 Hind III restriction sites. H. influenzae was once considered to be the cause of influenza until 1933, when the viral role of the flu became apparent; Most strains of H. influenzae are opportunistic pathogens – they usually live in their host without causing disease, but cause problems only when other factors (such as a viral infection or reduced immune function) create an opportunity. COS Site – is the Cohesive Site where lambda DNA naturally splits to “linearize” or “cyclize” itself; it’s about 12 overlapping nucleotides in length that results in “sticky ends”. Once inserted into a host cell, the COS sites base pair to cyclize the DNA. This helps prevent degradation via exonucleases. However, in the virus the DNA is linear. Cos sequences are ~200 base pairs long and essential for packaging. 

Hind III Digest of Lambda DNA 7 Restriction sites 8 DNA fragments Hind II Cuts at: A/AGCT T T TCGA/A

Agarose Gel Electrophoresis Theory & principles like PAGE. Samples include: MW markers (“DNA Ladder”). Two concentrations of genomic DNA extract prepared from leaves. Uncut  DNA.  DNA cut with Hind III. Caution: Agarose gels contain Ethidium Bromide. Intercalating agent, mutagen. Base pair insertions, deletions. Fluoresces pink (under UV light) in the presence of DNA. Caution: UV light causes cataracts & sunburns.

EZ - Vision DNA Stain Active Ingredient = 4',6-diamidino-2-phenylindole (aka “DAPI”). Binds to AT-rich regions of minor groove. Fluoresces blue under UV light. “Safer” than ethidium bromide. Bovine pulmonary artery endothelial cells stained with DAPI

Interpretation of Results Mainly . . . Estimate the amount of DNA in 1 g of plant tissue. For the DNA ladder components, make a graph of Rf vs. log10(no. of base pairs). Use to estimate/confirm fragment sizes of Hind III digest of  DNA. Speculate on differences in bp’s or absence of fragments. Note – our agarose gels are normally 1% (not 0.7% as shown in above gel).

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