Theoretical and practical methodology for the use of the polymerase chain reaction (PCR): Strategies for optimizing long-PCR performance including amplification of long fragments of DNA.

We begin by extracting DNA from tissues by digesting the tissue. Then we force the DNA to cling to a column, allowing us to wash away other parts of the cell. Once the other parts of the cell are washed away, we can collect the purified DNA.

Long PCR is a way of taking long pieces of extracted DNA (over 10,000 base pairs) and amplifying (copying) them using cycles of heating and cooling. We can start with a single strand of DNA, heat it to 98ºC causing the double helix to break, cool to 45-60º (in our class we used 55ºC) allowing primers (synthetic DNA templates around 20 base pairs) to attach onto the DNA at the site of a gene, heating up to 72ºC to allow enzymes to start connecting nucleotides to each DNA strand, finishing with a product of two double helixes of that specific gene. This can be repeated over many cycles, doubling the DNA each time, until we end up with 34 billion or more copies.

The copies of DNA can then be imaged by running them through gel electrophoresis, allowing us to separate molecules by their size. We also run a reference (1kb, or 1000 base pairs, for long PCR products, and 100bp for short PCR products) so we can compare the size of the known to the size of our PCR products. This provides a method of analyzing our product.

Strawberries make good subjects for at-home DNA extraction. They are octoploid, meaning they have 8 copies of their DNA. When the DNA is precipitated, having such a large quanity makes the DNA easy to see with the naked eye. We also extracted DNA from kiwi and banana.

You can download the instructions [.doc] and do this experiment at home.

Macherey-Nagel provided us with DNA extraction kits for this course. The kits allowed us to digest tissues and wash away the cellular contents, enabling the isolation and purification of DNA.

Prep: The DNA is a macromolecule stored inside of the mitochondria and nuclei of cells, which are organized into tissues. The first step of accessing the DNA is to dissect our salmanders to collect a sample of tissue. We used the liver of the coastal giant salamander, Dicamptodon ensatus, and the rough-skinned newt, Taricha granulosa. We chose the liver as liver cells contain a large number of mitochondria, as many as 200 mitochondria per cell.

Digest: We start to digest the tissue with enzymes, breaking it apart.

Lyse: to access the DNA, we have to open up the cells and then nuclei and mitochondria by lysing them.

Adjust: we adjust the DNA binding conditions by adding ethanol.

Bind: We then pipette the contents of our eppendorf tube into a column over another eppendorf tube. The column has silica beads which the negatively-charged DNA will stick to.

Wash: Washing the column with ethanol will wash most of the pigments and cellular contents away. We centrifuge to push the ethanol and its solvated contents through the column.

Wash x2: We move the column into a new eppendorf tube and wash and centrifuge again to remove more pigment and cellular contents.

Dry: centrifuge to remove remaining ethanol.

Elute: after moving the column to a new eppendorf tube, dH₂O is poured into the column washing the DNA with it.

Second elution: We performed a second elution to see how much DNA was remaining from the first. This elution contained even purer DNA, as the first elution still washed some pigments and cellular contents.

We also used some DNA extraction kits from Bio-Rad to extract DNA from Arabidopsis. These kits followed mostly the same procedure.

One large difference was that the cellular walls of the plants provided a bigger barrier, requiring us to finely mince our samples. Then a proprietary salt solution was used to open the cells instead of digesting them with enzymes.

PCR is the breaking apart of DNA double helixes (denaturation), the combining of primers to specific parts of a DNA strand (annealing), and the adding of nucleotides to the primer by polymerase (extension), allowing you to double the DNA at a specific location (in our case, a specific gene) in one thermal cycle.

1: Denaturation is the breaking of the hydrogen bonds that hold DNA strands together, forming the double helix. Hydrogen bonds are very strong, an electronegative element covalently bonded to a hydrogen will 'hog' the electrons, creating a dipole. The exposed proton (hydrogen) has a strong attractive force to electronegative elements. This can be broken with heat. We use 98ºC as it's enough heat to break the bonds, but low enough temperature that the enzyme (polymerase) won't denature.

2: Annealing is the act of joining a primer to the DNA strand. A primer is a synthetically-derived string of nucleotides (A, G, T, C bases) can be used as a template. At an appropriate temperature, that template will only fit onto the DNA strand at the specific sequence of nucleotides. We can bond, or anneal, the primer to a sequence of nucleotides just before a gene, allowing us to amplify (copy) that gene. Annealing is an art, not a science, as different temperatures will permit the annealing to occur at innappropriate sites. Annealing typically occurs between 45º and 60ºC. If the temperature is too cold, the primer can stick anywhere. If the temperature is too hot, it won't stick at all. We can predict appropriate temperatures based on the sequence, as A-T bonds contain two hydrogen bonds, and G-C contain three hydrogen bonds. This gives us information about how much energy is required to make the primer stick to the original DNA strand.

3a: Extension (short) is the lengthening of our primer. After we make the primer stick, we can use an enyzme that will walk along the bonded DNA and primer, adding appropriate nucleotides to the primer. DNA has direction, much as humans understand left and right. DNA is extended (nucleotides are added) in the 5' to 3' direction (five prime to three prime). Enzymes require very specific temperatures to function, and our DNA polymerase works at a predictable speed at 72ºC.

3b: Extension (long) is when we let our polymerase add enzymes for longer than 30-60 seconds. We can end up with copies of DNA 10,000 base pairs long or longer.

By using specific sequences in the DNA, we can anneal our primers to target points, allowing us to amplify a specific gene.

PCR generally goes for 25-35 cycles. You can figure out how many copies you have made using 2ⁿ where n is the number of cycles. For this class we used 35 cycles, yielding a calculation of 2³⁵ (34 billion) copies of each original strand of DNA (at the site of the gene selected by the primer). The cycles are continuous, in the above graph we have isolated a single thermal cycle.

Extension can last for variable time. The polymerase enzyme we used adds about 1kp (1000 base pairs, ie. the A, G, T and C nucleotides) per 30 seconds. Thus by letting the cycle run for 5 minutes, we are creating 10kb fragments of DNA. Manipulating the time for extension allows us to customize our gene fragment lengths.

In addition to adding the DNA to the PCR plate well, we have to make a small broth of chemicals (PCR mix) to make our PCR function.

Buffer: provides a suitable environment (pH, etc.) for PCR.

MgCl₂: polymerase requires specific orientation of the nucleotides during extension, the Mg²⁺ ion helps

dNTP: the A, G, T and C nucleotides which polymerase can grab to extend the primer

Primer 1: A sequence (in this case, 20bp long) that attaches just before a gene

Primer 2: A sequence (in this case, 20bp long) that attaches right after a gene.

Polymerase: The enzyme which attaches dNPTs to the primer

DNA template: The strand of denatured DNA we extracted from an organism, which the primer attaches to, allowing us to extend in the 5' to 3' direction creating a complementary DNA strand

Gel electrophoresis is a way of separating molecules by size. DNA fragments are negatively charged, and by creating a charge gradient the DNA molecules will move in the direction of positive charge. By adding an agarose gel, we can allow smaller molecules to move faster and bigger molecules to move slower.

A well is filled with a ladder, a reference material of known size. For our class we used ladders of 100bp or 1kb. The 100bp ladders contained molecules 100bp, 200bp, 300bp, 400bp and 500bp long. These move towards the positive end just like our DNA samples, creating a reference point of the size of our PCR products. This way we can make sure we have the right gene, as the gene will be of known length.

A 1% agarose gel is made by combining 0.5g agarose with 1 L of water, heating in a microwave until it just begins to boil, once the solution stops steaming 1 mL ethidium bromide (to stain) is added. While still very hot, this solution is poured into the mold inside a gel rig, and allowed to set. A comb is added, which creates wells (holes) for us to inject our ladder and 5 samples into. This will have six total lanes of molecules moving towards the positive charge.

We also need to create 1 L of 1x TAE (TRIS Acetate EDTA) buffer from a 50x concentrated solution. We know that (concentration₁)×(volume₁) = (concentration₂)×(volume₂), so we plug in our values of (50)×(X mL) = (1)×(1000mL) and solve for X mL, giving us 20 mL of 50x buffer needed to make a 1x solution. The 20 mL subtracted from our final solution volume of 1000 mL means we need 980 mL of distilled H₂O.

Adding a little buffer, we can remove our comb. The gel rig mold is then aligned, and the rig is filled with our 1x buffer solution. We add the ladder to the sixth well on the right, then the remaining five wells are filled with our PCR products.

A current is then applied, and after 1 hour and 15 minutes we have seperated our genes created in PCR and can compare them to the reference ladder for size, ensuring we have the right genes.

We can remove our gels, photograph them, and print images for our lab notebooks.

If you have any further questions or comments, feel free to contact Bob or Clay!