Genetic Engineering and Human Health

Polymerase Chain Reaction

In many criminal cases, a relatively small amount of DNA is collected at the scene of the crime, thus methods for amplifying or making many copies of the collected DNA is necessary for effective analysis. Before the process of Polymerase Chain Reaction (PCR) was invented by Kary Mullis in 1983, methods for amplifying a genetic sequence were timely and labor intensive. Thus, the discovery of the PCR allowed for a faster method of amplifying minute quantities of a DNA sample, which has revolutionized crime scene analysis. The PCR process includes a series of cycles in which the purified DNA sample is denatured, annealed with primers, and extended at the targeted sequence. After each cycle, the newly synthesized copies, called amplicons, become the template for the next cycle. Thus, within a few short hours millions of copies of the original small sample of DNA are available for better viewing or for storing. The amount can be quantified by the formula 2 ⁿ where n = the number of cycles. ²³ An advantage of PCR over other methods of amplification is that very little DNA is needed (~ 5-100µl), though most protocols for PCR ask for ~ 20-50µl. ²⁴ Today, the availability of PCR kits has allowed for forensic scientists to simply add their DNA sample for amplification.

Though commercial kits allow laboratories to simplify the preparation of targeted DNA, students need to first be familiar with the idea of targeting a sequence with restriction enzymes. A discussion of each step of the polymerase chain reaction is will follow a lesson in restriction enzymes. My students can follow laboratory protocols as long as they are able to practice the skills repeatedly, however when asked to verbalize their understanding of "why" they are doing what they are doing a gap in their knowledge becomes evident. Before students are allowed to purify their DNA samples, and run them through the thermal cycler, they will need to understand DNA extraction, denaturation of DNA, annealing of primers, and extension with nucleotides. In the end, students will have to predict the expected results of the gel electrophoresis of their amplified samples.

DNA extraction

Before DNA is amplified in a thermal cycler, the DNA must be extracted from the collected sample. To amplify a sample of DNA that has been collected from a crime scene several steps need to be taken to separate DNA from the cell. In essence DNA extraction means the cell membrane and nucleus need to be broken to expose the DNA. The trick is to break the cell's membranes without destroying the DNA. This can be accomplished through several different extraction methods. The use of Chelex beads will be discussed since that method will be used in my forensic science class.

Chelex, created by the Bio-Rad company, is a resin that is added to a sample of DNA. In effect Chelex adds magnesium ions which deactivate DNA nuclease which would otherwise digest the DNA strand for re-use as free nucleotides. ²⁵ Before the addition of Chelex, heat is applied to the sample to open the cells and separate the bonded strands. Chelex resins are negatively charged and help to remove positive metal ions. In order to prevent DNA nucleases from becoming activated, Chelex resins bind Mg+ ions thus preventing the nucleases from being activated. ²⁶ In doing so, nucleases are prevented from degrading the DNA strand. After the sample is centrifuged, purified DNA can be removed from the supernatant since the Chelex resin is forced to the bottom of the centrifuge tubes during centrifugation.

Other methods of extracting DNA include the use of organic chemicals (phenol-chloroform), specialized cellulose paper called FTA™, and differential extraction. All achieve the same purpose; however the Chelex method for DNA extraction is ideal for the classroom, because it does not require hazardous chemicals like the organic method. FTA™ paper is ideal for running multiple samples on an automated robotic workstation and once processed samples can be stored for multiple years. ²⁷

The thermal cycler is an important tool in performing PCR analysis. The thermal cycler performs the series of heating and cooling steps that allows DNA to denature, anneal, and extend with each cycle. Typically PCR amplification in thermal cycler will run for 25-30 cycles.

Denaturation of DNA

Recalling that hydrogen bonds hold DNA strands together, this bond is easily broken. Increase in heat will break the weak hydrogen bond causing the double helix to open or denature. Denaturation of DNA requires the sample to be heated above 90 ^?C. This does not break the bonds between the phosphate and deoxyribose sugar because they are covalently bonded, but interrupts the hydrogen bonds.

Annealing of Primers

The annealing process is the second step in PCR. Addition of primers "activates" free nucleotides to begin the DNA polymerase-mediated extension process. ²⁸ When they bind to DNA, primers act as signals, or starting points, for the action of DNA polymerase. One primer will bind upstream and the second downstream thus flanking the DNA strand. The primers are added in a 5' to 3' direction and the free nucleotides are added in the same manner. This annealing process occurs at a lower temperature, usually between 40 ^?C to 65 ^?C.

Commercially purchased PCR kits contain primers that serve to target the genetic sequence to be amplified. Primers are synthetically engineered and specifically designed to amplify a particular genetic sequence. Today many companies manufacture primers that are designed for a specific sequence. Scientists and laboratories can find catalogs on the World Wide Web for their target sequence. Primers are listed according to their genetic sequence, allowing scientist to choose the appropriate primer for their PCR samples.

Elongation

Addition of free nucleotides is controlled by the presence of a DNA polymerase that is thermally stable. ²⁹ Taq polymerase is a commonly used polymerase because its bacterium of origin (Thermus aquaticus) is a thermophilic organism found in hot springs.

The advantages of PCR include the relatively short amount of time it takes to amplify a specific gene of interest. Another is that a small sample of DNA is needed (as little as a single cell!), and crude preparations such as blood, semen, or saliva can be processed. On the other hand, PCR's limitations include false positives due to contamination with DNA from those handling the samples. It is also limited by the targeted gene sequence it is amplifying.

Conceptually PCR is easy to understand, but there are things that can go wrong in the process. Preparation of the PCR samples involves pipetting small quantities of DNA, primer, master mix, and purified DNA, which can lead to errors, either in the quantity moved from one tube to another or in contamination between samples. PCR tubes are tiny, holding only .2 ml. When exposed to open air, there is a risk of the sample evaporating, since it is often only ~ 20-50µl. Another problem that may arise is the creation of primer dimers. This occurs when primers bind before amplification. This premature binding of primers results in primer dimers which tend to be amplified more than the target sequence since they are only a few nucleotides in length as compared to the length of target sequence.

To prove that a DNA sample is being amplified and the "master mix" of primers, nucleotides and polymerase are all working, positive and negative controls are amplified along with the samples. Once complete, samples are loaded into gel boxes for electrophoretic separation.