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Biochemistry Laboratory DNA I (REVISED 4/02 KRD)
Purpose: Isolation of DNA from Gambusia liver.
The following general information is from: "Biochemical Techniques Theory and Practice" by
J.F. Robyt and B.J. White, Waveland Press Inc., 1987.
A. PREPARATION OF NUCLEIC ACIDS
1. General Separation and Purification Methodology
Nucleic acids are the most polar of the biopolymers and are therefore soluble in polar
solvents and precipitated by nonpolar solvents. In prokaryotes, DNA is double stranded and
circular and is found throughout the cytoplasm. In eukaryotes, DNA is located in the nucleus and
in mitochondria or chloroplasts. The DNA in the nucleus is double stranded and linear, whereas
the DNA in mitochondria and chloroplasts is like prokaryotic DNA, double stranded and
circular. The DNA in prokaryotes is relatively free of associated protein, but the DNA in the
nucleus of eukaryotes is associated with basic proteins, called histones.
Contaminating molecules that must be removed from both prokaroytic and eukaroytic
DNA are proteins and RNA. Proteins are denatured by the addition of organic solvents and
detergents, and RNA is removed with a brief treatment with deoxyribonuclease-free
ribonuclease. The high molecular weight DNA is soluble in the
aqueous phase, from which it is obtained by cold alcohol precipitation. Care must be taken
during precipitation, since high molecular weight DNA is readily sheared because of its long,
fibrous tertiary structure.As the alcohol is added, the DNA is carefully
"spooled" onto a glass rod as a threadlike precipitate.
RNA has less secondary structure and is of lower molecular weight than DNA. RNA is
single stranded and its secondary structure is intramolecular rather than intermolecular. There are
three types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA
(tRNA). Messenger RNA is found in ribosomes and free in the cytoplasm, where it is susceptible
to nucleases; mRNA molecules are variable in size. Ribosomal RNA, found associated with
proteins in the ribosome, is a relatively stable substance, and its molecules vary in size. Transfer
RNA is the smallest of the
RNAs and is distributed throughout the cell and organelles.
The N-glycosidic purine and pyrimidine linkages of DNA and RNA are stable to mild
acidic and basic conditions. However, the phosphodiester linkages of RNA are cleaved at 37oC
in 0.3 M KOH, resulting in the formation of 2'- and 3'-phosphoribonucleotides. The primary
structure of DNA is stable under these conditions. In dilute acid (0.1 N TCA, HCl, or HClO4)
nucleic acids will precipitate. In stronger acid and at higher temperatures (1N, 100oC, 15 min)
the purine bases of DNA are hydrolyzed from the 2-deoxyribose moiety. The pyrimidine bases
are hydrolyzed only under extreme conditions of pH and temperature.
Many of the nucleases present in cells can digest nucleic acids. When the cell is disrupted, the
nucleases can cause extensive hydrolysis. Nucleases apparently present on human fingertips are
notorious for causing spurious degradation of nucleic acids during purification.
The isolation and purification of DNA consists of four steps: (a) the disruption of cells
and membrane-bound structures to release DNA, (b) the inactivation of enzymes that
hydrolyze the DNA, (c) the dissociation and denaturation of protein, and (d) the solvent
extraction and concentration of the DNA by precipitation. Cells
are broken by grinding, tissue homogenization, or treatment with lysozyme (see Section 8.2).
Chelating agents are added to remove metal ions required for nuclease activity. DNA-protein
interactions are disrupted with SDS, phenol, and organic solvents. The DNA remains in the
aqueous phase, from which it is precipitated with cold (0oC) ethanol. The precipitate is usually
redissolved in buffer and treated with phenol or organic solvent
to remove the last traces of protein, followed by reprecipitation with cold ethanol. RNA is
removed by limited treatment with deoxyribonuclease-free ribonuclease.
RNA purification begins with a solvent extraction that denatures proteins and inactivates
cellular ribonucleases. Cell pastes or tissue homogenates are treated with phenol-saturated buffer
at 4oC. The aqueous and the phenol phases are separated by
centrifugation. The RNA containing aqueous phase is re-extracted with phenol-buffer, and the
various types of RNA in the aqueous phase are precipitated with alcohol and salt. Messenger
RNA is precipitated in 0.1 M NaCI and 70% ethanol, whereas rRNA is
precipitated in 3 M sodium acetate and 70% ethanol in which the small RNAs (tRNA and 5SRNA) are soluble, and tRNA is precipitated in 1 M NaCl and cold 66% ethanol.
B. PROTOCOL – Week 1
DNA ISOLATION
1. Gambusia dissection
a. remove the livers from 10 Gambusia.
b. place in a 1.5 ml eppendorf tube
c. grind tissue in 500 ul extraction buffer with a plastic pestle.
2. Proteinase K digestion
a. add 2 ul of [10 mg/ml] proteinase K
b. incubate at 65oC for 30 min
c. proteinase K is an enzyme that breaks down proteins
3. DNA extraction
a. add 500 ul of PCI (phenol:chloroform:isoamyl alcohol (25:24:1) - PCI extracts
and strips proteins bound to DNA
b. vortex for 1 min and centrifuge for 5 min in a table top microcentrifuge.
c. keep supernatant, don't take any of the material at the interface, yield is not
important.
d. add 500 ul of CI (chloroform:isoamyl alcohol 24:1) - CI removes excess phenol
e. vortex for 1 min and centrifuge for 5 min
f. keep supernatant, don't take any of the interface, yield is not important
g. add 500 ul of isopropanol to the supernatant to precipitate DNA. Do not mix! A
ropy precipitate should appear immediately. This is DNA.
h. Try to spool the DNA out of the tube with the tip of a Pasteur pipet and transfer to
a new Eppendorf tube.
i. Add 500 ul of ice cold 70% ethanol, finger vortex, and centrifuge for 5 min in
microcentrifuge at top speed. Remove supernatant, dry pellet for 10 min in speed
vac, and resuspend in 30 ul TE buffer.
NOTES:
* isopropanol only precipitates dsDNA; ssRNA stays in solution.
**If a ropy precipitate is not seen immediately, incubate at room temperature for
30 min (not colder because RNA will come out of solution)
j. centrifuge for 15 min in the microcentrifuge at top speed.
k. throw out supernatant and dry pellet in speed vac for 10 min.
l. resuspend in 30 ul TE Buffer.
4. Treatment of Isolated DNA with RNAse.
The DNA extraction procedure isolates both RNA and DNA. A hydrolytic enzyme
know as RNAse is used to remove RNA from a DNA solution. This enzyme hydrolyses
the phosphodiester bonds between ribonucleotides, but not deoxyribonucleotides.
a. Split your 30 ul sample into two 15 ul samples and treat one 15 ul sample with
RAse as described below:
Sample
RNAse (10 mg/ml)
15 ul
2 ul
b. Incubate 15 min at 37oC.
c. Add 100 ul of TE buffer.
d. Extract the sample with 100 up of PCI by vortexing for 1 min and then
centrifuging for 2 min.
e. Remove the aqueous layer (upper layer) to a new tube without disturbing the
interface (don't be greedy).
f. Add 15 ul of 3 M NaAcetate and 200 ul of ice cold 100% ethanol. Give to the TA
to freeze. Centrifuge for 10 min.
g. Remove the supernatant, air dry the pellet and resuspend in 15 ul of TE.
This should represent the amount of DNA in your sample. Read the absorbance of the
DNA in the spectrophotometer as described below.
5. UV Spectrophotometry of isolated DNA.
Read the concentration of your total nucleic acid solution first and then the concentration of
the DNA only solution after RNAse treatment when it is available.
a. Take 1 ul of fish DNA and add to 1.0 ml of TE Buffer.
b. Read in a quartz cuvette in the UV spectrophotometer.
c. Run a spectrum from 230 nm to 300 nm. Record the values for
230 nm
260 nm absorption peak of DNA
280 nm absorption peak of protein
300 nm "clear channel" gives light scattering and other nonspecific absorption value
6. Calculations for PCR:
a. Estimate the DNA concentration :
A260 = 1.0 = 50 ug/ml dsDNA
b. Estimate the purity of the DNA
A260/A280 = 1.9 for pure DNA. Contamination with protein will lower this ratio.
c. Estimate the specificity of your measurements.
A300 < 0.1 x A260. The absorbance at 300 nm should be less than 10% of the
absorbance at 260 nm.
7. PCR AMPLIFICATION
You will prepare your sample for PCR amplification by accurately determining the amount of
DNA in your sample. The rest of the PCR will be done by the TA.
--------------------------------------------------------------------------------DNA will be amplified using primers that amplify the cytochrome b gene in the mitochondrial
DNA. The amplified DNA will be loaded onto a 2.0% agarose gel
along with molecular weight standards to verify that the amplification did take place.
PCR amplification involves changing the temperature of the reaction for several cycles. The
DNA polymerase enzyme that is used was isolated from thermophilic bacteria and is thus
thermostable at temperatures as high as 95oC. DNA is denatured
at 95oC for 1 min. The temperature then drops to 52oC for another min to allow the primer to
anneal to the DNA. Finally, the temperature goes up to 72oC for amplification of DNA. These
temperature changes are repeated for 30 cycles resulting in the exponential amplification of
DNA.
Reaction recipe: Total volume of reaction 50 ul Master Mix: This mix will be provided in a tube
for each group.
10x buffer
MgCl2
dNTP*
Primer
Taq enzyme
5.0 ul
5.0 ul
10.0 ul (1 mM)
5.0 ul forward primer (10 uM)
5.0 ul reverse primer (10 uM)
0.125 ul (5 U/ul)
Total vol =
30 ul
Add:
DNA (100ng)
distilled H20
DNA vol.
Grand total
vol. necessary for 100 ng of DNA sample.
add to make total volume of 20 ul with DNA.
=
=
20 ul
50 ul
8. PCR Program
Stage 1
Stage 2
95oC for 3 min to denature DNA
30 cycles of:
95oC 1 min.
52oC 1 min.
72oC 1 min.
Stage 3
Stage 4
72oC for 10 min to finish any partial copies.
4oC until tubes are removed from machine.
The amplification will take approximately 3 hours, so you will analyze the amplified DNA next
week.
DNA database information about the cytochrome b gene from Gambusia affinis or Gambusia
holbrooki can be found at http://www.ncbi.nlm.nih.gov.
Search for cytochrome b sequence data by typing “Gambusia” in the search window (without the
“”).
Bring a printout of the cytochrome b sequence for Gambusia to class for next weeks lab.