Most of the greatest inventions; be it the first pacemaker or the penicillin which saved the world, all has its own incredible stories. Same is a case with the invention of Polymerase Chain Reaction (PCR). It is a device invented by Kary Bank Mullis for which he received the Nobel Prize. PCR was capable of amplifying or copying a very small amount of DNA fragment into large quantities.
The idea of creating PCR strangely struck me while I was driving to my home in California for the weekend. -Kary Mullis
The Story- Crazy Mullis
Mullis was born on 28th Dec 1944 in North Carolina USA and within few decades he was about to become one of the controversial figures among the scientific community. He likes his women, surfing and has also tried LSD and the initial thoughts were, “How could 1000 micrograms – one-thousandth of a gram – of some chemical cause my entire fu***g sensorium to undergo such incredible changes?” Defending astrology and questioning the science establishments were few of his activities which made him famous among other average Nobel laureates from chemistry.
Mullis received his bachelor’s degree in chemistry from Georgia Institute of Technology. He was an undisciplined and free-spirited boy throughout his schooling days. He completed his Ph.D. in Biochemistry from the University of California and after receiving the degree he abandoned his research career and started writing fiction.
Soon after the realization of his limited writing talents, he became a postdoctoral fellow at the University of Kansas Medical School. After working from 1973 to 1976, he returned to California for his second fellowship in pharmaceutical chemistry at the University of California.
In 1980, he left the university and started working as a biochemist at Cetus Corporation, Emeryville for synthesizing oligonucleotide probes. K. Mullis worked for 7 years on these probes which are short stretches of single-stranded DNA. In these 7 years, his road for PCR invention began. However, it would have not been accomplished without some of the previous works done by Watson, Crick, Kornberg, Har Gobind Khorana, Sanger, etc. Their discoveries like the structure of DNA, DNA polymerase (an enzyme responsible for DNA replication), DNA amplification by using DNA oligonucleotides, DNA sequencing and many more set the stage for PCR which K. Mullis utilized effectively.
It was the year 1983 when he was successful in conceptualizing the PCR and soon it became scientific folklore. In 1993, K. Mullis received the Nobel Prize for the same. This whole concept was considered as one of the monumental scientific techniques of the 20th century which led to various possibilities in sundry of fields. It has a multitude of applications in medicines, genetics, biotechnology, and forensics. Additionally, it also gave rise to the whole new discipline called paleobiology, wherein, DNA extracted from fossils is studied.
I love a microphone and a big crowd; I’m an entertainer, I guess. – Kary Bank Mullis
In 1986, he became the director at molecular biology department of Xyntronyx and in 1988 he started freelancing as a consultant. Along with Nobel Prize, he was also rewarded with several awards among which Japan Prize and Thomas A. Edison Award were the few. In 1998 he has written his own autobiographical book titled Dancing Naked in the Mine Field with him being on the cover with surf boat. Subsequently, he began providing expert pieces of advice in legal cases involving DNA and gave lectures at colleges and academic meets around the globe.
PCR- a Concept which made Kary Mullis Famous
Undoubtedly, PCR became one of the remarkable discoveries of modern molecular biology which made the completion of the Human Genome Project possible. From identifying infectious diseases to organ transplantation, its presence and necessity can be felt everywhere and is now the essential pillar of genomic science.
PCR: its Principle, Materials Required and the Process
It is based on the principle of enzymatic replication of nucleic acid which is used for quick and simple amplification of DNA sequences. These sequences can be obtained from different sources like skin saliva, semen, hair, blood and others. It only requires trace amounts of DNA for producing a large number of its copies making it as a sensitive assay.
DNA template, DNA polymerase, Nucleotides and Primers are the essential components required for the Polymerase Chain Reaction. The DNA template is the known DNA which is amplified and it can range from 100 – 1000 base pairs. DNA polymerase is the key enzyme used for replicating template strand by linking nucleotides together to form the product. The nucleotides are the building block of a DNA i.e. its four bases- adenine, thymine, cytosine and guanine (A, T, G and C). Primers are the artificial short single strand of DNA which matches the beginning and the end of the template strand. These serve as a starting point for DNA synthesis as DNA polymerase can add nucleotides only to the existing strand. Commonly, 18-22 base pair long primers are used.
To initiate the PCR process, DNA is obtained from the clinical sample and is mixed in the reaction mixture containing primers, nucleotides, polymerase enzyme, buffer, cation (MgCl2) and salts in a test tube or 96-well plate. Once mixed then it is placed in a thermal cycler which performs continuous cycles of DNA amplification in 3 basic steps i.e. DNA denaturation, primary annealing and extension of the primed DNA sequence.
- DNA denaturation- First, the mixture is heated to 94°C for separating the double DNA strands into two single strands.
- Primary annealing or hybridization- Temperature is lowered to 50–58°C for binding of primers with the target DNA segment at a specific site. Primers only bind if they are complementary to the sequence of target DNA i.e. A binding to G.
- Extension- The temperature is raised to 72°C at which DNA polymerase enzyme extends the complementary strand from the primers end by adding nucleotides. Generally, Taq polymerase is used due to its ability of efficient functioning at higher temperatures.
With each cycle of these 3 steps, the number of copied DNA molecules gets double and typically, the process is repeated 30 times which results in the 230 molecules of the desired product.
Analysis of the Resultant Product
There are 2 major methods which are used for analysis or visualization of PCR products called amplicons. The first and the simplest method is to stain the amplicons with ethidium bromide (a chemical dye) and then visualize the agarose gel containing the product under ultraviolet light; whereas, the second method involves labeling of primers or nucleotides with fluorophores before the PCR reaction.
The first method is the most widely used method wherein, PCR products are differentiated on the basis of size and charge on an agarose gel electrophoresis. These amplicons are run simultaneously with the predetermined set of DNA products with known sizes as a standard to compare the size of the resultant amplicons. The second method allows the fluorophores to be directly incorporated into the PCR product.
Types of PCR
- Nested PCR
- RT-PCR or Reverse Transcriptase PCR
- Real-Time PCR
- Gradient PCR
- Multiplex PCR
- AFLP PCR
- Allele-Specific PCR
- Assembly PCR
- Asymmetric PCR
- Colony PCR
- Hot Start PCR
- Inverse PCR
- In Situ PCR
- ISSR PCR
- Long PCR
- Single Cell PCR
- Standard PCR
Advantages of PCR
Its foremost advantages include greater sensitivity, fast analysis, ease of quantification, precision, reproducibility, good quality and least contamination. Apart from precisely identifying bacterial strains of divergent phenotypes, it can also measure a large number of samples at a time.
By using PCR we can amplify DNA or RNA by using just 1-100 cells or 0.1 µl of blood or cells scraped from buccal mucosa for excellent detection limits. Multiplex PCR allows us to search for different organisms or genes in one go and Nested PCR can detect the presence of low levels of bacterial DNA. RT-PCR is a useful and sensitive method for the detection of viruses.
Applications of PCR
PCR can be used for detecting and characterizing different microorganisms. Clinicians and researchers use PCR for diagnosing various diseases, cloning and sequencing genes. It helps in carrying out quantitative and genomic studies in a sensitive way.
It lets you pick the piece of DNA you’re interested in and have as much of it as you want. – Kary Mullis
PCR’s incredible ability to analyze infections, microbes, protozoa and others had a tremendous effect in reducing illnesses. It can also identify numerous pathogens like HIV causing AIDS, Hepatitis B and C, Human Papillomavirus, Neisseria gonorrhoeae, Cytomegalovirus and the rest.
PCR can be utilized in horticulture and sustenance businesses. It can also help in solving criminal investigations by identifying criminals as per the DNA obtained from the blood, hair, semen and so on. PCR can be used for DNA fingerprinting to solve parental disputes.
PCR is an essential and sensitive tool for tissue typing and organ transplantation. It can be used to detect genetic mutations or genetic disorders and can be utilized to build up connections among different species.
It played an important role in the completion of the Human Genome Project and can be beneficial for studying different life forms. It can also be utilized in paleontology for amplifying minute preserved DNA of extinct species or cryopreserved fossils dating back to millions of years from now.
After doing all the research on K. Mullis and PCR, there were few things which really intrigued me. Apart from his use of LSD, questioning the science and believing in astrology; he was also labeled as an undisciplined and free-spirited boy who was about to change the molecular biology forever. His different activities like surfing, writing and of course, his love for women makes me think that does he ever got distracted from his road to discovering PCR? And, who really is Kary Bank Mullis? A surfer; a writer; a free-spirited human; a Nobel Laureate; or All! Coming to the Polymerase Chain Reaction or PCR; indeed, the stage was already set by numerous scientists but what really inspired K. Mullis to invent it? And the most important questions of all, are there the only advantages of PCR? What about the drawbacks?
For Further Reading