Biohacking: PCR Primer

· November 17, 2012

Interested in biohacking but don’t know where to start? Do It Yourself  Polymerase Chain Reaction (PCR) is one easiest, most useful, and affordable biohacking technologies you can get started with.

PCR devices such as OpenPCR are available and on the market for just a few hundred dollars. Start here and you will be up and running, isolating and amplifying DNA samples in no time.

 

 

 

The Origin and Use of the Polymerase Chain Reaction

The ability to analyze, sequence, manipulate, and amplify specific DNA sequences is central to molecular biology. However the amount of DNA and RNA within cells is enormous; if stretched out end-to-end the DNA within each human cell would measure ~2.3 meters. Since long DNA molecules are very fragile, cellular DNA preparations are usually a complex mix of randomly broken DNA with many different sequences. Usually a scientist is interested in one gene or a few DNA sequences within this complex mix. Thus a major difficulty in molecular biology research has been how to isolate and amplify specific DNA sequences out of extremely complex mixtures. In 1983 this problem was solved by what has been called “the most important new scientific technology in the last 100 years” – the polymerase chain reaction or PCR. Here the discovery and uses of PCR will be briefly discussed.

DNA is a relatively simple polymer consisting of a sugar-phosphate “backbone” and four bound nucleotides (adenine [A], cytosine [C], guanine [G], thymine [T]) that normally pairs with another DNA molecule, forming a double helix. The nucleotides occupy the center DNA molecule with A binding to T, and G with C. This nucleotide pairing is extremely specific and DNA will preferentially form double-stranded molecules where the nucleotide binding matches perfectly, i.e., where A:T and G:C match along the entire double helix. RNA has a similar structure to DNA, but is often single stranded, shorter, and less stable than DNA. Double-stranded DNA normally becomes temporarily single stranded during DNA replication and RNA synthesis. It can also become single-stranded with increased heat (“heat denaturation”) and become double-stranded again with cooling (“renaturation/re-annealing”). Due to the high specificity of the nucleotide interactions renatured DNA usually forms a double helix with a perfectly matched DNA sequence. Additionally, DNA replication requires a “primer” or short double-stranded area for replication initiation. The requirement of “priming” for DNA replication and heat denatution and renaturation of DNA are central to PCR.

DNA is transcribed into different RNA types that carry the DNA sequence information. Many RNAs function to in protein synthesis, with the RNA nucleotide sequence determining the protein amino acid sequence, protein, and function. Although “DNA -> RNA -> protein” has been termed “the central dogma of molecular biology”, only about 1-2% of human DNA codes for protein, the remaining 98% either has no function (called “junk DNA”), or has as yet unknown functions. Since up to 93% of the human genome is transcribed into RNA at a low rate, much of this DNA likely performs poorly understood regulatory functions.

For many years the only way to isolate and amplify a DNA sequence was to cut the DNA at specific sequences with “restriction endonucleases”, place the DNA into a circular DNA molecular called a “plasmid”, and amplify the DNA by growing plasmid within bacteria. This process takes weeks and is labor intensive. A solution to this problem was found on a Friday night in April 1983 Dr. Kary Mullis was driving on US 101 in Northern California. Dr. Mullis was thinking about a proposed DNA sequencing experiment. During this drive it occurred to him that if double stranded DNA was heat denatured it could be re-annealed in the small priming DNA sequences called oligonucleotides. The oligonucleotides would act as primers to allow DNA synthesis if the appropriate enzyme and nucleotide precursors were present. If the primers were close together and on opposite strands of DNA, multiple cycles of denaturation and re-annealing would allow a section of DNA to be made over and over again. Each time the DNA is synthesized, the amount of the amplified sequence would double resulting in exponential growth. The steps are summarized below:

DNA Synthesis – New DNA is in blue and orange

1. Denaturation: The DNA sequences are heated to very high temperatures which reduces the DNA to individual strands. These individual strands then become accessible to bind with the primers.

2. Re-Annealing: During annealing the reaction mixture is cooled. The primers anneal to the complementary regions in the DNA  strands forming new double strands between primers and complementary sequences.

3. Synthesis: The DNA polymerase synthesizes a new complementary strand.

This entire process is repeated.

 

LSD, DNA, and PCR

By repeating the above steps a DNA sequence can be amplified up to 100 billion times within a few hours. The technology is so efficient that the DNA from a single cell can be amplified without difficulty. Thus what once took weeks to months to accomplish can be done in one afternoon with PCR technology. Dr. Mullis received the 1993 Nobel Prize in chemistry for the discovery of PCR. Interestingly, Dr. Mullis has used LSD and has stated that he doubted he would have invented PCR without his previous use of LSD. Dr. Mullis also claims to have had an encounter with an extraterrestrial in the form of a fluorescent raccoon, though not while under the influence of LSD.

 

Applications

The applications of PCR technology are many and include:

 1. Isolation and amplification of specific DNA sequences. With the correct primers nearly any DNA sequence can be isolated from a complex mix of DNA. Very high amounts of a specific sequence can be created even if there was originally very little in a DNA mix. With large quantities if a specific DNA sequence the DNA can be used for cloning, sequence and mutation analysis, and many other applications.

 2. Quantification of the amount of a specific DNA sequence within a complex DNA mix. Often the amount of a DNA or RNA sequence in a tissue sample allows one to diagnose the presence and severity of a disease, or the effectiveness of anti-cancer therapy. Many human cancers express bizarre gene fusions unique to the cancer. The presence of the “fusion genes” and their level can be very important in cancer management and treatment.

 3. Introducing changes into a DNA sequence. Using primers with small changes in their DNA sequence it is possible make DNA with a slightly changed/mutated sequence. The mutated DNA can be used to study the effects of the mutation in many systems – within cells or transgenic animals for example.

 4. Genomic amplification. PCR is vital for most types of DNA sequencing. Most DNA sequencing requires a relatively large amount of identical DNA, something not often found in complex DNA mixtures. PCR has been used to amplify entire genomes, including the human genome for later sequencing. Interestingly, the complete genomes of many extinct animals have been fully sequenced including the mammoth and Neanderthal genomes. Eventually it might be possible to bring back extinct species using PCR and other biotechnologies.

PCR has many variations that allow it to be adapted to many applications. The temperature of the reaction can be increased to make the DNA amplification more or less accurate. Less accurate PCR can identify DNA sequences related to the one amplified, revealing for example genes with similar sequences. Variations on different primers can be used to identify methylated DNA sequences and thus study epigenetic changes in specific genes. Multiple primers can be used to increase PCR accuracy in amplifying very low concentration DNA sequences (called “multiplex PCR”). The number of PCR variations numbers in the hundreds, making this technology central to molecular biology.

Also it’s interesting that the basic technologies to performed PCR where available for roughly fifteen years before Dr. Mullis thought of it during a long drive home. Thus PCR technology is an example of one insight radically changing an entire field of research. Without PCR much of the scientific progress in molecular biology over the past twenty years would not have been possible.

The key to what PCR amplifies is the sequences of the primers used in the PCR reaction. Primers are usually very short, 20-35 nucleotide long single-stranded DNA. Any primer sequence can be synthesized in automated DNA synthesizers. Since PCR is also a comparatively cheap technology, some Transhumanists and others have stated that “biohacking” will soon be possible, where individuals will be able to clone, manipulate, and use genetic information and gene sequences for their own use – much like computer hackers presently do. Presently now it is possible to “home brew” a genetic manipulation that allows one to produce glowing bacteria. The possible downside to this is obvious in bioterrorism. However those writing about the possibilities of biohacking emphasize that biohacking in not bioterrorism and its possible benefits include synthetic biology and possibly the eventual control over ones own genome. Presently altering ones own genome is impossible and the possible development of such technology and its intelligent application is in the distant future.


Looking for more information?

 

Biopunk and Biohacking Status Update

http://hplusmagazine.com/2012/10/05/biopunk-and-biohacking-status-update/

University of Utah Genetics Learning Center: PCR

http://learn.genetics.utah.edu/content/labs/pcr/

 

PCR and DNA Fingerprinting

 

OpenPCR Identification of Brussel Sprouts Haters

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