Exercise #4A - Isolation of Recombinant Plasmid DNA and Agarose Gel
Electrophoresis.
I. Overview:
Genetic engineering or recombinant DNA research is a very powerful investigative tool that has revolutionized the way in which cellular processes are studied in the laboratory. It is now possible to isolate specific fragments of DNA containing almost any gene from the vast amount of DNA comprising the genome of an organism. Genes are now mapped and characterized using physical techniques rather than genetic (recombinational) analysis. More important for the study of cell structure and function, the amino acid sequence of specific proteins can be deduced from the nucleotide code of the respective gene. Specific mutations can then be introduced into defined regions of a gene. Reintroduction of the modified gene back into cells by microinjection or transformation can often reveal much about the processes of gene regulation, and often the function of the protein itself. Underlying all procedures in recombinant DNA research is the principle of molecular or DNA cloning. This is the process by which a specific DNA fragment from a complex genome is introduced into an autonomously replicating genetic element such as a virus or a plasmid, and then amplified by propagation in a rapidly growing host cell. In this exercise, you will isolate plasmid DNA from a strain of E.coli bacteria. The plasmid you will isolate is a specially designed cloning vector that contains a DNA fragment derived from a human heat shock protein gene. You will characterize the plasmid DNA you isolate with respect to RNA and chromosomal DNA contamination using agarose gel electrophoresis. The purity of your preparation will be assayed by spectrophotometric and fluorimetric procedures. In the next exercise (Exercise 4B), you will characterize the human DNA fragment contained in the plasmid you have isolated will be physically mapped by restriction endonuclease digestion.
II. Background:
A. Bacterial Plasmids as Cloning Vectors: A typical molecular cloning experiment requires: (1) the DNA of inserted, often called foreign or passenger DNA; (2) a cloning vector; (3) restriction endonucleases; (4) DNA ligase; and (5) a prokaryotic or eukaryotic cell to serve as the biological host. Once the foreign and vector DNA have been isolated, they are treated with the same restriction endonucleases to introduce site specific cuts in the DNA. Many restriction enzymes produce double stranded cuts separated by four base pairs giving 5' or 3' single stranded extensions. The resulting DNA fragments will contain complementary single stranded ends, which can anneal to produce a recombinant molecule. The new molecules are made permanent only after all gaps are covalently sealed by DNA ligase. Once this has been accomplished, the recombinant molecules can be physically inserted into the appropriate host cell by transformation of microinjection. As the plasmid vector propagates in the dividing host cell, the foreign DNA is replicated and amplified as well. Virtually unlimited quantities of the recombinant molecule can be produced in this manner, which allow for physical characterization or manipulation of the passenger DNA fragment.
The molecular cloning vector plays an important role in recombinant DNA experiments. Since most DNA fragments are incapable of self-replication, particularly in a host cell from a different organism, an autonomously replication segment of DNA must be used to allow replication of the desired DNA fragment. Most cloning vectors have been derived from extrachromosomal replicons such as bacteriophages, viruses, and plasmids. Plasmids are small double stranded circular DNA molecules that are capable of replicating in bacteria independently of the bacterial chromosome. They contain a number of genes that confer a selective advantage to the host bacteria under certain growth conditions, but are not essential for bacterial propagation. Such gene products include antibiotic resistance factors, restriction endonuclease/host modification systems (which confer resistance to certain types of viruses), anti-bacterial factors (colicins) and occasionally metabolic enzymes that allow the use of specific compounds as nutrients. Most plasmid cloning vectors in use today were originally derived from a small number of R-factors (plasmids encoding antibiotic resistance). Thus, when carried by a host cell, they confer resistance to specific antibiotics. This property allows for the selective growth of host cells containing the plasmid. Thus, in a typical transformation experiment where less than 1 out of 106 cells actually take up plasmid DNA, only the cells containing the plasmid will be able to grow in the presence of the antibiotic. Other properties engineered into plasmid cloning vectors are cassettes of unique restriction endonuclease cleavage sites called multiple cloning sites and systems that allow host cells containing inserted DNA to be distinguished from cells containing the vector alone. The plasmid vector used in this exercise, BS/KS, has many other properties engineered into it that are beyond the scope of this course. Only those properties relative to this exercise will be described.
B. Plasmid BLUESCRIPT/KS: BLUESCRIPT/KS (BS/KS) is a small plasmid (about 3000 base pairs in length or 3 kilobasepairs [3 kb]) that replicates to a very high copy number in E. coli host cells. This plasmid contains a gene for resistance to the drug ampicillin, a penicillin derivative that normally kills E. coli. The plasmid also contains a multiple cloning site containing recognition sequences for 21 different restriction enzymes. The multiple cloning site is located within a gene encoding beta galactosidase. Using a host stain that is lac-, BS/KS transformed bacterial colonies containing foreign DNA inserts can be distinguished from those containing the vector without an insert. Colonies containing the vector alone are lac+ and have a blue color in the presence of the indicator X-gal. Insertion of a foreign DNA fragment into the beta galactosidase gene disrupts its function, giving a lac- colony that is white in the presence of X-gal.
Like all plasmids, BS/KS exists in the form of a covalently closed circle of double stranded DNA. It does not integrated into the host cell chromosome but contains its own origin of replication. Plasmid DNA is present in multiple forms in the cell, however, which can be resolved by gel electrophoresis. In rec- host strains that are defective in genetic recombination, most of the plasmid DNA exists as a supercoiled monomer of 3 kb. A small amount of plasmid is present in the form of a 6 kb dimer and a fraction exists as a "nicked" or open circle. In the latter form, one strand of the DNA has been cleaved by an endonuclease. This allows the supercoiled DNA to unwind or "relax". In wild type rec+ hosts, much of the plasmid DNA exists as higher order multiples of the unit length monomers (ie. dimers, trimers, tetramers, etc.).
C. Isolation of Plasmid DNA: Small covalently closed circular DNA is much more resistant to denaturation by high pH or elevated temperature than linear DNA. That is the principle behind two simple and rapid techniques used to isolate relatively pure plasmid DNA. In a procedure developed by Holms and Quigly, cells are briefly treated with the enzyme lysozyme to weaken the cell walls and then heated at 100o for 2 min in the presence of a non-ionic detergent. The cell membrane is dissolved by the detergent and most cellular proteins are denatured and precipitated by the high temperature. Chromosomal DNA is broken into large fragments during cell lysis and rapidly becomes single stranded at 100o. When the sample is cooled on ice, the denatured chromosomal DNA aggregates into a gel, while the supercoiled plasmid DNA remains in solution. A brief centrifugation pellets the protein and denatured chromosomal DNA and the plasmid DNA (contaminated with RNA) is recovered from the supernatant by alcohol precipitation.
A second procedure that was developed by Birnboim and Doty, takes a little
longer, but is more reproducible and yields DNA that can more readily cut with
restriction enzymes. Cells are lysed by an alkaline SDS solution at low
temperature. The sheared genomic DNA molecules released during lysis become
single stranded while the covalently closed circular plasmid DNA does not. The
solution is then made acidic by the addition of potassium acetate at low pH.
This causes the denatured genomic DNA to aggregate into an insoluble gel. In
addition, most of the cellular protein also precipitates as insoluble
potassium-dodecyl sulfate complexes. Residual proteins are removed by phenol
extraction and the plasmid DNA along with most of the RNA is recovered by
ethanol precipitation. DNA prepared this way can be cut by most restriction
enzymes although more enzyme is usually needed to cleave a given amount of this
crude DNA than with more highly purified preparations. RNase must be included
in the digestions in order to degrade the contaminating RNA which would
otherwise obscure small restriction fragments. You will be using the alkaline
lysis procedure in this exercise.
D. Agarose Gel Electrophoresis:
Nucleic acids are routinely separated according to their mass and structural
properties by electrophoresis on either agarose or polyacrylamide gels. Double
stranded molecules, such as covalently closed circular plasmid DNA move more
rapidly through the gel matrix than open circles or linear DNA molecules.
Linear double stranded DNA molecules have a very uniform structure in solution
and a constant negative charge per unit length resulting from the phosphate
backbone of the helix. Thus, they migrate towards the anode during
electrophoresis at a rate that is inversely proportional to the log of the
molecular mass (in either daltons or base pairs). The molecular mass of double
stranded DNA fragments can be determined after electrophoresis by comparison
with standards of known molecular masses (just like proteins run on SDS gel).
The resolving power of a gel depends upon the concentration of matrix material
used, which in turn determines the pore size of the gel. For example, a 2%
agarose gel can resolve double stranded DNA molecules as small as 300 base
pairs (bp). For large DNA molecules, a low concentration of agarose is used
(0.3 - 0.4%). Polyacrylamide gels are "tighter" than agarose gels and
can be used to separate DNA fragments between 6 (20% polyacrylamide) and 1000
(3% polyacrylamide) bp in length. A limiting factor is the consistency of the
gel itself. Gels with too high a matrix concentration (>2% agarose or
>20% polyacrylamide) are too rigid to handle. Similarly, gels will low
matrix concentration (<0.3% agarose or <2.2% polyacrylamide) are too
sloppy to handle easily. Agarose or polyacrylamide gels can also be used to separate
single stranded DNA and RNA. However, denaturing electrophoresis conditions are
usually used (urea, alkaline pH, or other denaturants) in order to eliminate
differences in secondary structure. Under denaturing conditions, single
stranded nucleic acids separate according to molecular weight.
Horizontal agarose gels, often called submarine gels, are routinely used in the
laboratory to analyze DNA. Molten agarose is simple poured into a tray and
allowed to solidify after inserting a comb to for the sample wells. The gel is
then place into an electrophoresis chamber in a horizontal orientation and
covered with running buffer. The buffer and gel form a current bridge between
the two electrode chambers. Samples of DNA in a dye buffer solution containing
a dense solution of glycerol of Ficoll are then layered in the wells. Following
electrophoresis, DNA is visualized by staining with the fluorescent dye
ethidium bromide (a probable carcinogen!), which causes the DNA bands to
fluoresce when illuminated by UV light (also carcinogenic!). Ethidium bromide
can be included in the gel during the run in order to speed things up. Gels are
recorded by photography using a Polaroid camera with a UV filter.
III. Procedure:
A. Isolation of Vector and Recombinant Plasmid DNA:
1. Obtain from your instructor 1.4 ml of bacterial culture (E.
coli) containing BS/KS vector and another 1.4 ml of culture containing
KS/BS with an unknown DNA insert. Note the number of the unknown you are given.
2. Collect the cells by centrifuge for 30 sec in an Eppendorf microcentrifuge.
3. Remove the medium by aspiration and try and leave the bacterial pellets as
dry as possible.
4. Resuspend the pellets by vortexing in 100 ul of ice cold Solution I containing:
50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0. It is critical that the cells
are completely dispersed in this solution, so vortex thoroughly.
5. Store for 5 min at room temperature. The top of the tubes need not be closed during this step.
6. Add 200 ul of a freshly prepared solution of 0.2 N NaOH, 1% SDS (Solution
II).
7. Close the top of the tube and mix the contents by inverting the tube
rapidly three times. Do not vortex. Store the tube on ice for 5 min. If you
exceed this time, or allow the sample to warm up, the preparation will contain
a large amount of irreversibly denatured plasmid DNA which migrates slightly
faster than the supercoiled form on agarose gels and cannot be cut with restriction
enzymes.
8. Add 150 ul of ice cold Solution III, which is made up as
follows: To 60 ml of 5 M potassium acetate, add 11.5 ml of glacial acetic acid
and 28.5 ml of distilled water. The resulting solution is 3 M with respect to
potassium and 5 M with respect to acetate. Close the cap of each tube and vortex
gently in an inverted position for 10 sec. Store on ice for 5 min.
9. Centrifuge for 5 min to sediment the white precipitate containing KDS,
protein, and chromosomal DNA.
10. Transfer 400 ul of each supernatant to a fresh correctly labeled tube.
Alternatively, decant the entire supernatant into a fresh tube.
11. Add 400 ul of 1:1 phenol/chloroform. It is ok to add the phenol/chloroform
to the tube ahead of time. Vortex for 10 sec and centrifuge for 2 min. 12.
Transfer the upper phases to a fresh tubes, being careful to avoid bringing
over any of the denatured protein from the organic interface.
13. Add two volumes of ethanol at room temperature. Mix and let stand at
room temperature for 2 min.
14. Centrifuge for 5 minutes to collect the nucleic acid
precipitate.
15. Remove the supernatants. Stand the tubes in an inverted position over a
paper towel to allow all of the fluid to drain away.
16. Carefully add about 1 ml of 70% ethanol at room temperature. Gently
"tumble" the tubes 3 times. Let stand 5 minutes. "Tumble"
the tubes 3 more times. This will rinse away excess salts from the precipitate.
17. Again carefully remove all the supernatant. If the pellet re-suspends, centrifuge again. Dry the pellet for 10 min under vacuum.
18. Dissolve each pellet in 50 ul of TE buffer.
19. Label 6 tubes as follows: KS (control), KS + RNase, KS + DNase, R
(recombinant), R + RNase, R + DNase.
20. Pippet 5 ul of your KS plasmid to each of the tubes labeled KS. Pippete
5 ul of your recombinant plasmid (with the recombinant insert) to each of the
tubes labeled R.
21. Add 2 ul of RNase (200 ug/ml) to each tube labeled +RNase, and 2 ul of
DNase (100 ug/ml) to each tube labeled + DNase. Add 2 ul of TE buffer to the
remaining KS tubes.
22. Incubate all 6 tubes for 10 min at 37oC.
23. Add 2.5 ul of gel loading buffer to each of your samples for
electrophoresis.
24. Add 5 ul of RNase (200 ug/ml) to the remaining 35 ul of your KS and
Recombinant plasmid preparations. Incubate 10 min at 37oC to degrade
the RNA. Hold your samples on ice until you take samples for spectrophotometric
and fluorimetric analysis in part C below.
B. Electrophoretic Analysis of DNA Samples:
1. While the plasmid DNA is being prepared, one member of your
lab group should prepare a 1% agarose gel. Your instructor will provide you
with information about the size of the gel to cast and the amount of agarose
solution to use.
2. Obtain a gel tray of the appropriate size and seal the ends with autoclave tape
as directed by your instructor. If your gel apparatus uses ruber dams, the tap
is not necessary.
3. Place the tray on a level surface and pour the correct amount of molten
agarose (held in a 60o water bath) into the sealed tray. Avoid
bubbles. If bubbles occur, remove them with a Pasteur pipette before the gel
solidifies.
4. Introduce the sample well comb and remove any bubbles that adhere to the
teeth of the comb. Allow the gel to solidify for at least 30 min.
5. Remove the gel comb and place the gel into an electrophoresis chamber with
the wells closest to the cathode (-) end. Add enough running buffer to the
chamber to just cover the wells. Let your instructor check the buffer level
before you proceed.
6. Using a P-20, carefully underlay your samples into 6 adjacent wells. Load 5
ul of marker DNA in a 7th well.
7. Run your samples at 50 V until the tracking dye is within about 1 cm from
the bottom of the gel. (If you are running late, it is ok to stop the gel when
the dye is about half way)
8. Using gloves, remove the gel tray and place it on the UV transilluminator.
Put on protective glasses and view the gel with UV light. Photograph the gel to
record your results.
C. Spectrophotometric and Fluorimetric determination of RNA and DNA
Concentration.
1. Measure the absorbance of the total nucleic acids in your plasmid DNA
preparation at 260 nm by pipetting 5 ul of each sample into a spectrophotometer
cuvette containing 1 ml of TE buffer. Make sure the cuvette has been zeroed
first. Mix your sample by covering the cuvette with parafilm and inverting.
2. Measure the concentration of DNA in your sample using the spectrofluorimeter
as you did in Exercise #1. Use 2 ul of your sample, dilute if necessary.
(STORE YOUR REMAINING PLASMID DNA SAMPLES AT -20oC FOR USE IN LAB
4B)
3. Record the following:
a. The absorbency at 260 nm of 5 ul of your samples diluted into in 1 ml of TE.
b. The concentration of DNA in your samples (ug/ml).
4. Calculate the following:
a. The concentration of total nucleic acid in each of your samples.
b. The total amount of nucleic acid in your original samples (before you
removed 15 ul for gel analysis.
c. The total amount of DNA in each remaining sample (that is how much you have
left to digest in Lab 4B).
d. The total amount of RNA in each remaining sample.
e. The total amount of DNA originally extracted from the cells.
f. The total amount of RNA originally extracted from the cells.
IV. Final Evaluation:
Answer the following in the Discussion section of your lab report:
a. Why can the concentration of RNA still be measured after digestion with
RNase?
b. Is your plasmid DNA preparation contaminated with genomic (chromosomal) DNA?
If so, estimate the % of contamination.
c. How many forms of plasmid DNA are present in your preparation?
d. What is the apparent size of the covalently closed circular form of the BS/KS
plasmid? (calculate using semi-log paper) The molecular mass of the plasmid is actually ~3000 bp. Explain why
a supercoiled DNA molecule migrate so rapidly in an agarose gel.
References:
Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J.D. (1983) The Molecular Biology of the Cell. pp. 185-192. Garland, New York.
Birnboim, H. C. and Doty. (1979) Nucleic Acids Research. 7:1513
Holms, D. A. and Quigly, M. (1981) Analytical Biochemistry. 114:193
Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning. Cold Spring Harbor Laboratory