BiDil, A-HeFT, and the Future of Race in Pharmacogenetics

PCR (Polymerase Chain Reaction)

— What kind of natural process does PCR mimic?

PCR aims to mimic the DNA replication that occurs naturally during cell division.

Here are some differences:

1. Instead of the RNA primers that are used in natural DNA replication (http://www.ncc.gmu.edu/dna/replicat.htm), PRR uses short (about 20-30 base pairs), synthetic DNA strands (http://en.wikipedia.org/wiki/Primer_%28molecular_biology%29), called oligonucleotide primers (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.section.552). These primers are synthesized to target (by nucleotide matching) particular areas of interest in a portion of DNA (http://courses.washington.edu/hubio514/modules/topic5.html).

2. While RNA primers are attached to the denatured (separated) strand of DNA with RNA polyerase (gmu.edu), oligonucleotide primers attach to two denatured DNA strands by a temperature decrease of 50-70 degrees C (nih.gov).

The primary similarity in the processes is that both end with DNA polymerase elongation, which I'll detail below.

— What are the steps of PCR and what does each step accomplish?

Before the machine-automated cycles of PCR, several ingredients are mixed: DNA, amplimers, singular nucleotides, a chemical buffer that emulates the chemical environment of natural DNA replication, and DNA polymerase. Apparently this mixing can lead to incorrect nucleotide binding, so one technique used to overcome this is to place the ingredients in a medium that keeps them physically separate until the PCR cycles begin. One medium is wax, which then melts and allows true mixing. The use of pre-fabricated wax pellets containing the separated DNA and primer, which is added to a test tube with liquid polymerase and buffer, is called hot-start PCR (nih.gov).

The machine-automated cycles are:

1. Denaturing: The mixture is heated to 94-96 degrees C to break the hydrogen bonds that hold the two strands together. This takes 1-2 minutes, though the first cycle of automated PCR may be longer to ensure that the DNA and primers are single-strand only (http://en.wikipedia.org/wiki/PCR#Primers).

2. Annealing: The mixture is cooled to 50-70 degrees C to allow oligonucleotide primers to bind to the matching part of each DNA strand (http://nobelprize.org/chemistry/laureates/1993/illpres/pcr.html). Usually two oligonucleotide primers are included to define the start point and end point of interest on the DNA sequence. The oligonucleotide primers will bind to opposing DNA strands. One strand, called the leading strand, has an exposed 3' nucleotide carbon at one end and an exposed 5' nucleotide carbon at the other end. The other strand, called the lagging strand, has its exposed carbons reversed.

3. Elongation: DNA polymerase will complete each strand by using the loose nucleotides, adding complementary nucleotides to the remaining single bases on the strand. Elongation will start at the end of the primer with an exposed 5' nucleotide carbon. GMU calls this a 5'-3' synthesis, and wikipedia has a good chemical diagram of this at http://en.wikipedia.org/wiki/Nucleic_acid_nomenclature

One strand of DNA will go through about 20-30 PCR cycles (wikipedia). This will make more than a billion copies of one cell fragment (Desalle/Yudell 41).

— Did the simplicity of the PCR process surprise you? In what ways?

I was surprised, and in reading Kary Mullis's 1993 Nobel speech in which he describes his discovery of PCR (http://nobelprize.org/chemistry/laureates/1993/mullis-lecture.html), I was even more surprised by how surprised he was by the discovery. It happened in part by Mullis being in the right place at the right time: Working in a mind-numbing job killing lab rats at UCSF, unsure of what he wanted to do with his life (after earning his Ph. D. he tried to become a fiction writer and had worked for a time as a waiter), he stumbled on a lecture discussing gene cloning. This led to a new interest in DNA synthesis and a job working at Cetus, a company where a machine was "turning out oligonucleotides much faster than the molecular biologists at Cetus could use them" (Mullis's speech). It was this abundance that led Mullis to experiment with the oligonucleotides, which eventually led to PCR.

Perhaps because PCR is such a simple technique composed of several techniques already in existence, none of Mullis's friends or colleagues were excited by his early ideas for PCR. As Mullis says: "However, shocking to me, not one of my friends or colleagues would get excited over the potential for such a process... There was not a single unknown in the scheme. Every step involved had been done already. Everyone agreed that you could extend a primer on a DNA template, everyone knew you could melt double stranded DNA. Everyone agreed that what you could do once, you could do again."

It seems that when it comes to a 'revolutionary' scientific technique, it's not uncommon for its creator to borrow heavily from other scientists' research. It's also not uncommon for the full impact of the technique not to be felt for a long time. Both are true of PCR... but it's still surprising to me.

Even though the technique is simple and built on pre-existing techniques, it took a long time for Mullis to get it to work properly. Mullis's first test of PCR was to copy a 400 base pair portion of Human Nerve Growth factor-- and it didn't work. After months of failed PCR trials on human DNA, Mullis was had a successful trial on the DNA of a much simpler organism, pBR322, a purified plasmid.

The final thing that surprised me-- or at least resonated with me, since it's not really a surprise-- is Mullis's implication that the kind of childhood and college experimentation that shaped his scientific mind wouldn't necessarily be legal today-- while he grew up in a "golden age" of chemistry, today he'd be viewed as a "menace to society." In these times, with the increasing restrictions put on regular citizens and the increasing suspicion of amateur chemists (for instance, a member of the famous art group Critical Art Ensemble was arrested for having what amounts to a high school lab in his home), I wonder how it will influence the practice of science in the next generation.

— Why do you think PCR has been such a revolutionary technique for molecular biologists?

1. SPEED. DeSalle and Yudell state that before PCR, the progress of the Human Genome Project was slow because replication was only available with cell-based (in-vivo) cloning, which takes several weeks. PCR allows for test-tube-based (in-vitro) cloning, which is cheaper and much more rapid-- it only takes a few hours. Time is needed to design primers (nih.gov), but this process has been aided with the writing of primer-design computer software, several of which are listed at the Rosalind Franklin Center for Genomics Research (http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html).

2. EXPONENTIAL AMPLIFICATION. Each PCR cycle increases the PCR product by a base 2 exponent. For instance,

10 cycles yields 2 ^ 10 = 1024 copies.

20 cycles yields 2 ^ 20 = 1048576 copies.

30 cycles yields 2 ^ 30 = 1073751824 copies.

These yields are vastly higher than with in-vivo replication (http://people.ku.edu/~jbrown/pcr.html).

3. AUTOMATION. Because PCR consists of only a few short steps, it can be mechanized. This saves human time and effort, and prevents human error. These machines are inexpensive, so many labs have them and many different kinds of scientists have access to them. This page at SUNY Stonybrook is a PCR machine FAQ for all students, even beginning genetics undergrads! http://www.osa.sunysb.edu/dna/rtpcr_faq.html

4. NOT THE BE-ALL, END-ALL!! Because in-vivo cloning can render longer segments of DNA and can render them more accurately, PCR did not render in-vivo cloning obsolete. The DNA made by PCR alone is often too limited for study (nih.gov). Instead, PCR-produced DNA is often integrated into in-vivo systems and then cloned further (nih.gov).

— What are some possible applications of this technology?

This technology reminds me of photocopying an article at a library-- the researcher wants a personal copy that she can carry around and mark up, but the librarian wants the original article to remain in as good condition as possible. I'm also reminded of backing up a computer-- we make backups so that we can continue our work if the original data is lost. In large institutions, redundant backups are made-- many copies stored in different places for extra security. PCR, with its huge amplification yields, allows for this kind of redundancy.

PCR is used in many ways already:

1. GENETIC FINGERPRINTING. PCR's ability to exponentially amplify a tiny amount of DNA has made it a boon to criminal forensics; a tiny sample of human material from a crime scene (saliva, blood, semen) can yield enough DNA to be accurately compared with the DNA from a suspect. A genetic fingerprint is made by targeting areas of the DNA that are prone to "microsatellites" (http://en.wikipedia.org/wiki/Microsatellites) or VNTRs (variable number tandem repeats, http://www.dnai.org/romanovs/index.html). Microsatellites are short base pair sequences that are repeated over and over. The location and number or repetitions per location are almost entirely unique-- a person has a 1 in 5 million chance of having the same genetic fingerprint as another person.

2. MATERNITY/PATERNITY TESTING. A child will have a similar microsatellite pattern (location and repetitions per location) to a closely related relative like a father or mother. Again, PCR is used to amplify a portion of DNA and compare the microsatellite patterns of two people. In maternity testing, mitochondrial DNA can be used since it is passed to a child only through the mother (http://www.dnai.org/romanovs/index.html).

3. DETECTION OF HEREDITARY DISEASES. With diseases like cystic fibrosis and sickle cell anemia that are triggered by only a few mutant alleles, PCR is used to amplify a potentially mutated region, which is then saturated with allele-specific oligonucleotides that bind to the alleles. Because the region has been amplified, detection with oligonucleotide saturation is much simpler (http://courses.washington.edu/hubio514/modules/topic5.html).

4. DETECTION OF INFECTIOUS DISEASES. PCR's ability to largely amplify a tiny portion of DNA makes it helpful in detecting infectious diseases at early stages. A disease like tuberculosis is hard to detect in its early stages because while the infecting bacteria is present, few of them are pathological. Again, PCR will amplify a portion of DNA, making it easier to find the bacteria (http://courses.washington.edu/hubio514/modules/topic5.html).

4. ANALYSIS OF ANCIENT DNA. Any small portion of DNA can be amplified, including the DNA of long-dead creatures. PCR has been used to analyze wooly mammoth DNA, Egyptian mummy DNA, and the DNA of Anna Anderson, an old woman who claimed to be the daughter of a long-dead tzar (http://en.wikipedia.org/wiki/PCR, http://www.dnai.org/romanovs/index.html).

5. CLONING GENES. Isolating and cloning certain genes in simple organisms like the zebrafish, a favorite of geneticists because of its cleara embryo (http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/), has helped to identify important genes with similar functions in humans (http://courses.washington.edu/hubio514/modules/topic5.html).

6. MUTAGENESIS. This is a way of making a change to an organism's DNA's nucleotide sequence. Basically a portion of DNA is cloned using PCR, altered, and then brought into the source cell. NHGRI's biggest project involving mutagenesis is the Knockout Mouse Project, in which 10,000 (to date) mice have been produced, each with a different nullified gene. Observing a mouse without a particular gene, should, through a kind of process of elimination, help to clarify what the absent gene effects (http://www.genome.gov/Pages/About/RecentArticles/AustinKnockoutMouseCommentary.pdf).