Methods Nucleic Acids 1

Agarose Gel Electrophoresis PCR DNA Sequencing

Agarose gel electrophoresis

Support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Agarose gel electrophoresis is used to separate nucleic acid molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).

Agarose is purified from agar, a gelatinous substance isolated from seaweed. Different purities of agarose are commercially available as are agaroses with different melting properties. Agarose forms pores ranging from 100 nm to 300 nm in diameter depending upon the concentration of agarose used in the gel.

Agarose Gel Electrophoresis and Resolution limits

The DNA moves toward the positive anode due to the negative charges on its phosphate backbone. Small DNA strands move fast, large DNA strands move slowly through the gel as shorter molecules are less impeded by the pores than are longer molecules. Small DNA molecules experience little frictional drag from solvent and gel molecules and migrate rapidly. Larger DNA molecules encounter more friction and corrospondingly move at a slower speed. Although the most important factor is the length of the DNA molecule, conformation of DNA is also a factor. Usually linear molecules are separated on an agarose gel DNA fragments from a restriction digest, linear DNA PCR products, or RNAs. Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The higher the voltage, the faster the DNA migrates. But voltage is limited by the fact that it heats up the gel and beyond a point lowers resolution. The separation range is also affected by the electroendosmosis (EEO) value of the agarose, this being a measure of bound sulphate and pyruvate anions. The greater the EEO the slower the migration rate.

Small nucleic acids are better separated by polyacrylamide gels, large DNA molecules are only able to move end-on and are more difficult to separate.

Buffers
There are a number of buffers used for agarose electrophoresis. The most common being: Tris Acetate EDTA (TAE), Tris Borate EDTA (TBE) and Sodium Borate (SB). TAE has the lowest buffering capacity but provides good resolution for larger DNA gels have to be run at a lower voltage for a longer time. SB is relatively ineffective in resolving fragments larger than 5 kbp; however, with its low conductivity, a much higher voltage can be used, which means a shorter analysis time. TBE provides adequate buffering and good resolution and is most commonly used.

Visualization
The most common dye used for agarose gel electrophoresis is ethidium bromide, abbreviated as EtBr. The ethidium bromide intercalates between the DNA bases and fluoresces reddish-orange under ultraviolet light. By running DNA through an EtBr-treated gel and visualizing it with UV light, distinct bands of DNA become visible.

Preparation of Agarose Gels

The vast majority of agarose gels used are prepared and run horizontally. An appropriate amount of agarose powder is mixed in a buffer solution. The solution is brought to a boil to dissolve the agarose and then allowed to cool down to about 60 C; while cooling the solution is stirred or swirled. 5 l ethidium bromide stock is added per 100 ml gel solution and stirred to disperse the ethidium bromide. The molten gel is poured into a Perspex gel rack. A comb is inserted into one side of the gel, about 5-10 mm from the end of the gel to form wells for the samples. When the gel has set, samples are loaded into the wells and electrophoresis is carried out with the gel submerged under buffer in a tank. Apart from DNA, the loaded samples include glycerol and a tracking dye (eg. bromophenol blue, cresol red, xylene cyanol). Glycerol is used to weigh down the samples. The DNA is not visible during the run so a tracking dye is added to the DNA to avoid the DNA being run entirely off the gel. The tracking dye has a low molecular weight, and migrates faster than the DNA or at a specific rate, allowing tracking of DNA and preventing it running off the gel. After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a transilluminator, while using protective gear to limit exposure to ultraviolet radiation) to view the DNA bands. The ethidium bromide intercalates between the DNA bases and fluoresces reddish-orange under ultraviolet light.

Agarose gel with samples loaded in the slots, before the electrophoresis process

A pattern of DNA-bands under UV light

The electrophoretic mobility or distance traveled by a DNA fragment during electrophoresis is inversely proportional to the logarithm of its size (usually given as base pairs or kilobase pairs) The migration distance of a DNA fragment in question can be compared with a plot of the distances migrated by a set of standard nucleic acid fragments to determine its size.

In the given plot; a) How far will a 0.5 kb DNA fragment travel? b) What is the size of a fragment which has migrated 16 mm?

The relationship between the log of a DNAs size and its electrophoretic mobility deviates strongly from linearity if the DNA is very large.

In standard agarose gel electrophoresis, it is difficult to separate molecules more than 50 kb as longer molecules run as a single slowly migrating band. To separate them, orthogonal field alternation gel electrophoresis (OFAGE) is used. The electric field alternates between two pairs of electrodes each positioned at an angle of 45 to the length of the gel. DNA molecules still move down the gel but each change in the field forces the molecules to realign. Shorter molecules realign more quickly than longer ones and so migrate more rapidly through the gel. OFAGE enables molecules upto 2 Mb in length to be separated.

The Polymerase Chain Reaction (PCR)

In polymerase chain reaction (PCR), a DNA polymerase is used to amplify (ie. replicate) a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, ie., alternately heating and cooling the PCR sample to a defined series of temperature steps.

The Principles of PCR

In polymerase chain reaction, two primers are chosen/synthesized that have sequences complementing primer binding sites at the 3 ends of the gene in question. The strands are separated by heating to a temperature above the "melting temperature" of the double- or partially-double-stranded form; then the temperature is lowered allowing the two primers to anneal to the primer binding sites. A temperature-resistance polymerase Taq polymerase is used to catalyse growth from DNA primers. Primers on opposite strands are extended in different directions towards each other. After completion of the replication of the segment between the primers (one cycle), the two new duplexes are heat denatured to generate single stranded templates and the a second cycle of replication is carried out by lowering the temperature in presence of all the components needed for polymerization. Repeated cycles of synthesis and denaturation result in an exponential increase in the number of segments replicated (upto a millionfold ). Thus, a single copy of a human gene can be detected after amplification provided primers can be synthesized corresponding to known sequences of the gene.

The Polymerase Chain Reaction

Ingredients for PCR

1) Template DNA: Usually the amount of template DNA is in the range of 0.01-1 ng for plasmid or phage DNA and 0.1-1 g for genomic DNA. Higher amounts of template DNA may increase the yield of nonspecific PCR products. Even trace amounts of agents used in DNA purification procedures (phenol, EDTA, Proteinase K, etc.) strongly inhibit Taq DNA Polymerase. Ethanol precipitation of DNA and repetitive treatments of DNA pellets with 70% ethanol is usually effective in removing traces of contaminants from the DNA sample. 2) Primers: Appropriate primers need to be used. Primer concentrations should not go above 1M unless there is a high degree of degeneracy; 0.2M is sufficient for homologous primers. 3) MgCl2 Concentration: Taq requires free Mg2+. Since Mg2+ ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. The recommended range of MgCl2 concentration is 1-4 mM, under standard reaction conditions.

4) dNTPs: The concentration of each dNTP in the reaction mixture is usually 200 M. It is very important to have equal concentrations of each dNTP (dATP, dCTP, dGTP, dTTP), as inaccuracy in the concentration of even a single dNTP dramatically increases the misincorporation level. When maximum fidelity of the PCR process is crucial, the final dNTP concentration should be 10-50 M, since the fidelity of DNA synthesis is maximal in this concentration range. 5) Taq DNA Polymerase: Usually 1-1.5 u of Taq DNA Polymerase are used in 50 l of reaction mix. Higher Taq DNA Polymerase concentrations may cause synthesis of nonspecific products. However, if inhibitors are present in the reaction mix (e.g., if the template DNA used is not highly purified), higher amounts of Taq DNA Polymerase (2-3 u) may be necessary.

Thermophilic DNA polymerases

The thermophilic DNA polymerases, like other DNA polymerases, catalyze templatedirected synthesis of DNA from nucleotide triphosphates. A primer having a free 3' hydroxyl is required to initiate synthesis and magnesium ions are necessary. In general, they have maximal catalytic activity at 75 to 80C, and substantially reduced activity at lower temperatures. Eg. at 37C, Taq polymerase has only about 10% of its maximal activity. Taq DNA polymerase (Taq polymerase/ Taq) is a thermostable DNA polymerase first purified from the hot springs bacterium Thermus aquaticus in 1976. Taq's temperature optimum for activity is 75-80C with a halflife of 9 min at 97.5C. Taq can amplify a 1-kb strand of DNA in 30-60 seconds at 72C. Its main drawback is its lower replication fidelity as it lacks a 3 to 5' exonuclease proofreading activity. Pfu is slower than Taq and typically requires 2 minutes to amplify 1 kb of DNA at 72 C. It appears to have the lowest error rate of known thermophilic DNA polymerases.

Cycling Conditions
Initial Denaturation Step. The complete denaturation of the DNA template at the start of the PCR reaction is of importance. Incomplete denaturation of DNA results in the inefficient utilization of template in the first amplification cycle and in a poor yield of PCR product. The initial denaturation should be performed over an interval of 1-3 min at 95C if the GC content is 50% or less. This interval should be extended up to 10 min for GC-rich templates. If the initial denaturation is no longer than 3 min at 95C, Taq DNA Polymerase can be added into the initial reaction mixture. Denaturation Step. Usually denaturation for 0.5-2 min at 94-95C is sufficient, since the PCR product synthesized in the first amplification cycle is significantly shorter than the template DNA and is completely denatured under these conditions. Primer Annealing Step. Usually the optimal annealing temperature is 5C lower than the melting temperature of primer-template DNA duplex. Incubation for 0.5-2 min is usually sufficient. However, if nonspecific PCR products are obtained in addition to the expected product, the annealing temperature should be optimized by increasing it stepwise by 12C.

Extending Step. Usually the extending step is performed at 70-75C. The rate of DNA synthesis by Taq DNA Polymerase is highest at this temperature. Recommended extending time is 1 min for the synthesis of PCR fragments up to 2 kb. When larger DNA fragments are amplified, the extending time is usually increased by 1 min for each 1000 bp. Number of Cycles. The number of PCR cycles depends on the amount of template DNA in the reaction mix and on the expected yield of the PCR product. For less than 10 copies of template DNA, 40 cycles should be performed. If the initial quantity of template DNA is higher, 25-35 cycles are usually sufficient. Final Extending Step. After the last cycle, the samples are usually incubated at 72C for 5-15 min to fill-in the protruding ends of newly synthesized PCR products. Also, during this step, the terminal transferase activity of Taq DNA Polymerase adds extra A nucleotides to the 3'-ends of PCR products. If PCR fragments are to be cloned into T/A vectors, this step can be prolonged up to 30 min.

Applications of PCR
1) PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as Southern and Northern blotting and DNA cloning, that require large amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material. 2) PCR can also be used for the isolation of a DNA sequence for recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. 3) PCR may also be used for genetic fingerprinting, a forensic technique used to identify a person or organism by comparing experimental DNAs. As PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. A variation of this technique can also be used to determine evolutionary relationships among organisms. Thus, PCR may also be used in the analysis of ancient DNA that is thousands of years old. .

4) In analysis of diseases. Viral DNA can be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. 5) Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification

DNA sequencing

DNA sequencing by the Sanger procedure (Dideoxy sequencing)

The sequencing method most commonly in use now was developed by Fred Sanger. Dideoxynucleotides, which lack OH groups at the 3 as well as the 2 position of deoxyribose, are used to terminate DNA synthesis at specific bases. These molecules are incorporated normally into growing DNA strands. Because they lack a 3 OH, however, the next nucleotide cannot be added, so synthesis of that DNA strand terminates. After separating the DNA strands, DNA synthesis is initiated with a radioactive primer. Four separate reactions are carried out, each containing one dideoxynucleotide mixed with its normal counterpart as well as the three other normal deoxynucleotides. When the dideoxynucleotide is incorporated, DNA synthesis stops, so each reaction yields a series of products extending from the radioactive primer to the base substituted by a dideoxynucleotide. Products of the four reactions are separated by electrophoresis and analyzed by autoradiography to determine the DNA sequence. Comparison of bands produced by labelled fragments from the four tubes provides a display of successively shorter fragments when read from top to bottom. The appearance of the band in a particular lane indicates the dideoxy nucleotide incorporated. The sequence of the original strand is complementary to the sequence obtained.

Automated DNA sequencing

Four separate sequencing reactions are performed, each containing one chainterminating dideoxynucleotide and a primer labeled with a distinct fluorescent tag. The products are then pooled and subjected to gel electrophoresis. As the DNA strands migrate through the gel, they pass through a laser beam that excites the fluorescent label. The emitted light is detected by a photomultiplier, which is connected to a computer that collects and analyzes the data. Fluorescent detection is widely used in automated DNA sequencing machines.