Download Characterisation of DNA by Agarose Gel Electrophoresis and

Biochemistry Practical Course I
Characterisation of DNA by Agarose Gel Electrophoresis
and Melting Curves
1. Introduction
Deoxyribonucleic acids (DNA) are the carriers of the genetic material of the cell, i.e. they serve
both, the passing on of information to a new cell during mitosis as well as code for anabolic and
catabolic processes within the cell. From the fact alone that information about very complex
patterns of life are preserved in the DNA one may deduct that deoxyribonucleic acids are structures
of high molecular mass. The building blocks of DNA are the 2’-deoxyribonucleotides which consist
of a sugar moiety (de-oxyribose), a phosphoric acid residue and a base. Amongst the bases one can
differentiate between the purines (adenine and guanine) and the pyrimidines (thymine und
cytosine). Deoxyribose and phosphate residue are linked alternatively by 3’-5’-phosphodiester
bonds and play an important structural role as DNA backbone. The genetic information is put down
as the varying combinatorial possibilities of the base sequence. During duplication of the
chromosomes which precedes mitosis (cell division) the DNA is duplicated semi-conservatively.
For expression the information in the DNA is transferred to a messenger ribonucleic acid (mRNA)
in a process called transcription, and is subsequently translated into the corresponding protein
amino acid sequence during protein biosynthesis which is called translation. Contrary to the doublestranded DNA RNA is a single-stranded macromolecule where the deoxyribose is substituted by
ribose and the base thymine by uracil. The steric arrangement of the single nucleotides in the DNA
and its constituents (bases, de-oxyribose und phosphate residue) determines the physico-chemical
properties of the DNA in dependence on the surrounding milieu. According to Watson and Crick
the DNA molecule represents a double-helix where the hydrophilic sugar and phosphate residues
are arranged on the outside and the bases, stacked on top of each other like a spiral staircase, are
pointing to the inside of the molecule. The single strands of the double-helix are held together by
hydrogen bonds between the base pairs which are always formed by adenine with thymine and
guanine with cytosine. In dependence on the water and salt contents the DNA helix can exist in
different conformations. A-DNA und B-DNA helices are right-handed while Z-DNA has a lefthanded structure. Contrary to the DNA in prokaryotes eukaryotic DNA is arranged in the cell
nucleus. Additionally eukaryotic DNA is associated with basic proteins called histones. They take
an important role in causing the high condensation of DNA in its chromosomal form for transport
during cell division. Besides the DNA in the nucleus extrachromosomal DNA is found in
mitochondria and plasmids. Its origin is discussed in the theory of endosymbiosis. Bacteria usually
have a cyclic chromosome. Very often they contain additional genetic elements called plasmids.
Some plasmids enable their carrier to exchange genetic material by direct cell-to-cell contact
(conjugation). As the information for resistance to antibiotics is localized on the plasmids
conjugational processes take a decisive part in producing resistance towards antibiotics containing
drugs.
2. Characterization of DNA by restriction cleavage followed by agarose gel
electrophoresis
The aim of this experiment is to subject plasmid DNA which has been isolated in two different
ways to restriction endonuclease cleavage and analyze the results by gel electrophoresis.
This technique permits the determination of a DNA fragment’s size by comparison to a reference of
known nucleotide length (“marker”, fig. 1). The DNA bands in the gel are visualized by the dye
ethidium bromide, which emits fluorescence in the visible spectrum when it is bound to a DNA
molecule (fig. 2). The migration speed of a DNA fragment is dependent both on the mass and the
conformation of the molecule (figs. 3 & 4).
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Biochemistry Practical Course I
1)
2)
Fig. 1: Gene Ruler 1000 bp ladder (Fermentas).
Fig. 2: Intercalation of ethidium bromide in DNA.
3)
Fig.3: Theory of electrophoretic mobility (LOTTSPEICH).
4)
left: OGSTON-theory. DNA is described as a spherical molecule.
Molecules which are smaller than the pore size may diffuse almost unhindered. At increasing
volume of the species, collisions with the gel matrix will occur more often, and frictional forces slow
down the migration speed.
right: Reptation model. The molecules is aligned along the electric field and „wriggles“ through the
gel pores, reminiscent of a snake.
Fig. 4: Influence of conformation on migration speed.
Supercoiled DNA (form II) migrates faster than the „open form“ (circular DNA) ; the migration
speed of the linear form (form III) lies in between.
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Biochemistry Practical Course I
Schedule for restriction analysis
1.
2.
3.
4.
5.
Minipreps of the plasmid DNA from an overnight shaking culture by two methods
Restriction cleavage of plasmid DNA
Preparation of agarose gels for the electrophoretical separation of DNA fragments
Electrophoresis
Analysis of electrophoresis
2.1. Conventional plasmid miniprep.
1. Harvest 4 ml of an overnight shaking culture in table centrifuge (13,000 rpm/max speed, 30 sec)
2. Resuspend cell pellet in 100 µl ice-cold TEG.
3. Add 200 µl freshly prepared 0.2 N NaOH, 1% SDS (premix 150 µl 0.4 M NaOH and
150 µl 2% SDS: do not keep NaOH/SDS-mixture on ice!).
4. Gently mix by inverting the vessel several times. Allow cells to lyse: incubate 5 min on ice.
5. Add 150 µl ice-cold KAcO/HAcO, gently invert several times to mix.
6. Incubate 5 min on ice.
7. Spin in table centrifuge (13,000 rpm/max speed, 10 min).
8. Transfer supernatant into fresh eppendorf tube. Add 450 µl chloroform/isoamylalcohol to
supernatant. „Vortex“– please wear nitril gloves.
9. Spin in table centrifuge (13,000 rpm, 2 min) to separate the layers.
10. Transfer upper water-soluble phase carefully into fresh eppendorf tube, don’t transfer phase
boundary layer.
11. Repeat steps 8, 9 and 10 once again.
12. Mix upper phase (ca. 400 µl) with 2 Vol. of freezer-cold EtOH abs. (800 µl). Place the tube into
freezer at -20 C to precipitate plasmid DNA for 30 min period at least.
13. Spin in table centrifuge (13,000 rpm/max speed, 10 min), remove (avoid touching of DNA
pellet) and air-dry the pellet for 5-10 min.
14. Redissolve pellets in 25 µl water or elution buffer from the mini-prep. kit.
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2.2. Plasmid miniprep with kit (Plasmid DNA Purification, Macherey-Nagel)
1. Transfer overnight culture into a 2 ml Eppendorf-Cup
Centrifuge 30 sec. at 13000 rpm. Discard supernatant.
2. Resuspend cell pellet in 250 µl buffer A1 by “vortexing“ until completely suspended.
3. Add 250 µl buffer A2. Mix gently by inverting several times (do not “vortex”).
Incubate at room temperature for a maximum of 5 min.
4. Add 300 µl buffer A3, mix again by inverting several times (do not “vortex”).
Centrifuge 10 min at 13,000 rpm.
5. Place mini-column (Spin Column) in a 2 ml collecting tube.
6. Load the supernatant from step 4 onto the column.
Centrifuge 1 min at 13,000 rpm.
7. Remove mini-column, decant filtrate and put column back again.
Add 600 µl buffer A4 and centrifuge 1 min at 13,000 rpm.
8. Remove mini-column, decant filtrate and insert column back.
Add 600 µl buffer A4 and centrifuge 1 min at 13,000 rpm.
Discard filtrate and repeat centrifugation for 2 min at 13,000 rpm to remove residual washing
buffer.
9. Take out mini-column and place into a new 1.5 ml Eppendorf tube.
Add 75 µl H2O or elution buffer to the column, incubate 1 min at room temperature and
centrifuge again for 1 min to collect DNA.
10. The DNA concentration can be determined by OD260 on NanodropTM (optional step)
2.3. Restriction analysis of plasmid DNA
Plasmids can serve as vectors for insertion of DNA fragments. Desired DNA fragment can be
multiplied as part of the plasmid by transforming bacteria with plasmid and following growth of
bacterial culture. Desired fragment of DNA can be excised by restriction endonucleases. They are
enzymes of bacterial origin which specifically cleave double stranded DNA at precise cleavage
sites. In the practical course we use the enzymes BamH I (5´-G GATCC-3´) und Hind III (5´A AGCTT-3´). Resulted DNA fragments can be separated according to their size by gelelectrophoresis. Depending on the fragment’s size one can choose between agarose or
polyacrylamide gels for electrophoresis.
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Total volume of every cleavage reaction: 20 µl
1. Take 2 µl 10 x buffer for restriction enzymes
2. Take 4 µl or accordingly 14 µl from miniprep.
3. If necessary make up to 20 µl with H2O (Take into consideration the volume of the
restriction enzymes!)
4. Take 1 µl (ca. 5 U) restriction enzyme
5. Incubate for 1 hour at desired temperature (ref. restriction enzyme)
6. Terminate the digest by adding 4 µl DNA sample buffer (6 x)
7. Load onto 1% agarose gel and start electrophoresis (80-110 V)
DNA type
Sample #
Macherey-Nagel kit
chloroform extraction
2
3
4
5
6
7
without enzyme BamH I Hind III both both
without enzyme
14
14
14
14
4
14
DNA
2
2
2
2
2
2
10x buffer “R”
BamH I buffer
BamH I
Hind III
H2O (ad 20 µl)
4
1
1
3
3
all volumes in µl
Load samples on gel (1% agarose):
1: DNA–ladder (standard) 10 µl
2: without enzyme
3: BamH I
4: Hind III
5: both (kit)
6: both (kit, less amount)
7: without enzyme (chloroform extract)
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1
2
1
1
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Biochemistry Practical Course I
2.5. Solutions for minipreps
TEG-Buffer (100 ml)
50 mM
Glucose
25 mM
TRIS/HCl pH 8
10 mM
EDTA/pH 8
NaOH/SDS
0.4 N
NaOH
2%
SDS
Mix 1 : 1 (not on ice)
KAcO/HAcO
KAcO
Glacial acetic acid
H2O
0.91 g
Ethanol
Chloroform
Chloroform/isoamylalcohol
1.6 g/100 ml
2 g/100 ml
29.46 g
11.5 ml
Up to 100 ml
10 TAE-Buffer (1000 ml)
400 mM
TRIS/AcO pH 8
10 mM
EDTA
pH 8
Ethidium bromide
Stock solution: 10 mg/ml
Addition: 10 µl/100 ml gel
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Biochemistry Practical Course I
2.6. Vector
Figure 5: Schematical presentation of the expression vector pET from Novagen TM
5.4 kb from vector
862 bp from insert
total plasmid size – about 6.2 kb
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Biochemistry Practical Course I
3. Characterisation of DNA by melting curve analysis
The strands of a double-helix quickly dissociate when the hydrogen bonds between the base pairs
are loosened. This can be achieved by heating of DNA solution. The unwinding of the double-helix
is called melting and it occurs rather suddenly at the certain temperature. The melting temperature
(Tm-value) is defined as that temperature at which half of the helical structure is lost. The melting of
DNA can be easily detected by measuring the absorption at 260 nm. The dissociation process of the
base pairs brings about an increase in absorption. This is called the hyperchromic effect. Alteration
of secondary DNA structure upon rising of the temperature results in change of light absorption.
The consequence is a sigmoidal temperature dependence. The melting point coincides with the
inflection point of the sigmoidal curve. It strongly depends on the base composition in the studied
DNA. Large number of G-C base pairs increases Tm of DNA, while DNA with mainly A-T
composition has lower melting point. The nature of base pair interaction can serve as an explanation
for that phenomenon. G-C base pairs have three connecting hydrogen bonds compared to A-T base
pairs which have two hydrogen bonds only. Thus, breaking down of stronger G-C interactions
requires more energy. The melting point of a DNA sample hence gives information about its base
composition (G/C content).
3.1. Melting point determination of DNA samples
Sufficient nucleic acid solution is pipetted into cuvettes to achieve an extinction value between 0.1
and 0.2.
Solution A: 15 mM NaCl
1.5 mM Na-citrate
Solution B: 500 mM MgCl2
Samples for measurement:
a) For large cuvettes (2.5 ml):
1. Solution A is used as reference – 2500 µl
2. 25 µl of artificial poly-dA-dT in 2475 µl of solution A
3. 25 µl of the same artificial poly-dA-dT in 2225 µl solution A and 250 µl solution B
4. 15 µl of calf thymus DNA in 2485 µl of solution A
b) For half-micro cuvettes (1.5 ml):
1. Solution A is used as reference – 1500 µl
2. 15 µl of artificial poly-dA-dT in 1485 µl of solution A
3. 15 µl of the same artificial poly-dA-dT in 1335 µl solution A and 150 µl solution B
4. 10 µl of calf thymus DNA in 1490 µl of solution A
The cuvettes are placed in the heating cuvette holder. Absorption of each sample is read at 260nm
starting from initial 30 C. The absorbance of each sample is detected stepwise every 2 C upon
rising of temperature.
For the determination of Tm the absorption values are plotted against the corresponding
temperature. The inflection points of the sigmoidal curves give the melting point of your DNA
samples.
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The curves have to be normalised (see fig. 6) to compare different samples in one graph. Therefore,
the data has to be plotted the following way: observed change in absorption ( At) per maximally
achieved change in absorption ( Amax) against the corresponding temperature value. This is done
for each curve independently:
At
A max
A min
A min
At
A max
The minimum value is subtracted from all absorption values (baseline correction). All values are
then divided by the max. value; thus finally all normalised values are a percentage value of the max.
absorption.
3.2. Example of DNA melting curves
Figure 6: Melting curve of an unknown DNA species.
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