PCR (Polymerase Chain Reaction)

PCR

Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1984 by the American biochemist Kary Mullis at Cetus Corporation. It is fundamental to much of genetic testing including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics.

The majority of PCR methods rely on thermal cycling. Thermal cycling exposes reactants to repeated cycles of heating and cooling to permit different temperature-dependent reactions – specifically, DNA melting and enzyme-driven DNA replication. PCR employs two main reagents – primers (which are short single strand DNA fragments known as oligonucleotides that are a complementary sequence to the target DNA region) and a DNA polymerase. In the first step of PCR, the two strands of the DNA double helix are physically separated at a high temperature in a process called Nucleic acid denaturation. In the second step, the temperature is lowered and the primers bind to the complementary sequences of DNA. The two DNA strands then become templates for DNA polymerase to enzymatically assemble a new DNA strand from free nucleotides, the building blocks of DNA. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the original DNA template is exponentially amplified.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. If the polymerase used was heat-susceptible, it would denature under the high temperatures of the denaturation step. Before the use of Taq polymerase, DNA polymerase had to be manually added every cycle, which was a tedious and costly process.

Applications of the technique include DNA cloning for sequencing, gene cloning and manipulation, gene mutagenesis; construction of DNA-based phylogenies, or functional analysis of genes; diagnosis and monitoring of hereditary diseases; amplification of ancient DNA; analysis of genetic fingerprints for DNA profiling (for example, in forensic science and parentage testing); and detection of pathogens in nucleic acid tests for the diagnosis of infectious diseases.

CRISPRCas9

CRISPRCas9

CRISPR-Cas9 is a method of genome editing that exploits a natural DNA-snipping enzyme in bacteria, called Cas9 (CRISPR-associated protein 9) to target and edit particular genes. CRISPR stands for Clustered regularly interspaced short palindromic repeats, which are segments of DNA of a particular structure found widely in bacteria and archaea (prokaryotes). In the wild, the CRISPR-Cas9 system is part of the prokaryotic immune system, which can snip out of the genome DNA acquired from foreign sources such as phages (bacterial viruses). The same molecular machinery is now being used to enable genetic material to be cut from and pasted into the genomes of other organisms, including eukaryotes such as humans. It might offer a tool for curing genetically based diseases.

DNA has become a versatile polymeric substrate for making nanotechnological structures and artificial molecular-scale machinery for computation, pattern formation, and nanoscale assembly. For several decades now, these efforts have drawn on methods developed in and for biotechnology, and similarly they are likely to find ways of exploiting the advantages of the new technique called CRISPR/Cas9 for manipulating DNA. #CRISPRCas9

CRISPR-Cas9 is a method of genome editing that exploits a natural DNA-snipping enzyme in bacteria, called Cas9 (CRISPR-associated protein 9) to target and edit particular genes. CRISPR stands for Clustered regularly interspaced short palindromic repeats, which are segments of DNA of a particular structure found widely in bacteria and archaea (prokaryotes). In the wild, the CRISPR-Cas9 system is part of the prokaryotic immune system, which can snip out of the genome DNA acquired from foreign sources such as phages (bacterial viruses). The same molecular machinery is now being used to enable genetic material to be cut from and pasted into the genomes of other organisms, including eukaryotes such as humans. It might offer a tool for curing genetically based diseases.

DNA has become a versatile polymeric substrate for making nanotechnological structures and artificial molecular-scale machinery for computation, pattern formation, and nanoscale assembly. For several decades now, these efforts have drawn on methods developed in and for biotechnology, and similarly they are likely to find ways of exploiting the advantages of the new technique called CRISPR/Cas9 for manipulating DNA.

FASTER WAY TO PRODUCE SPECIFIC HUMAN ANTIBODIES USING NANOPARTICLES

Treating patient derived B cells with nanoparticles coated with CpG oligonucleotides to stimulate plasma cell production and challenge antigens to designate what kind of antibody the B cells should produce has resulted in the generation of specific, high affinity antibodies in just a few days that can recognize several strains of a pathogen at the same time. The researchers have already produced antibodies to a variety of bacterial and viral antigens, including tetanus toxoid and several strains of influenza, and were able to generate anti HIV antibodies from B cells donated by healthy volunteers who did not have the disease.

The new technique also eliminates the need for previous exposure to the pathogens, either by vaccination or infection.

"Our technique should allow the production of these antibodies within a shorter time frame in vitro and without the need for vaccination or blood/serum donation from recently infected or vaccinated individuals," said Dr. Facundo Batista, who led the team from the Francis Crick Institute in London, the Ragon Institute of Massachusetts General Hospital, MIT and Harvard. "In addition, our method offers the potential to accelerate the development of new vaccines by allowing the efficient evaluation of candidate target antigens."

Antibodies are produced by white blood cells called B cells, which recognize the calling card of a bacteria or virus called an antigen, and transform into plasma cells tailored to produce large numbers of antibodies to that specific antigen that fight off the disease. That's what happens in the body. When scientists tried to reproduce the process in the lab, they ran into problems getting the B cells to make the specific kind of plasma cells they needed because the challenge antigens were missing.

It was easy to get the B cells to proliferate by adding short DNA fragments called CpG oligonucleotides into their culture medium. CpG oligonucleotides activate a protein inside B cells TLR9, but TLR9 enthusiastically stimulates every B cell in the sample to respond, not just the tiny fraction capable of producing a particular antibody. Batista and his colleagues attached both CpG oligonucleotides and real challenge antigens to nanoparticles, added them to B cell cultures, and the plasma cells that resulted were both abundant and pathogen specific.

The team hopes their approach will help researchers produce therapeutic antibodies to treat infectious disease and other conditions, such as cancer.

For more information, go to the Journal of Experimental Medicine
http://jem.rupress.org/cgi/doi/10.1084/jem.20170633?PR
https://www.eurekalert.org/pub_releases/2017-07/rup-rdn071717.php

Yoshinori Ohsumi: Autophagy from begining to end

Yoshinori Ohsumi was influenced by his father, who was a professor of engineering at Kyushu University, He was familiar with academic life while he was growing up. But whereas his father worked in a very industrially oriented field, he was more interested in the natural sciences. In high school, he was interested in chemistry, so he entered the University of Tokyo to learn chemistry. He quickly discovered chemistry wasn't so attractive to him, because the field was already quite established. But he was lucky, he thinks, because the early 1960's was the golden age of molecular biology. He decided he wanted to work on that instead.

There were not very many molecular biology labs in Japan at that time. He joined Dr. Kazutomo Imahori's lab as a graduate student to study protein synthesis in E. coli. Unfortunately, he did not get very good results in his work, and, when he had finished his graduate studies, he discovered it was very difficult to find a good position in Japan. So, on Dr. Imahori's advice, he took a postdoctoral position with Dr. Gerald Edelman at The Rockefeller University in New York.

As a graduate student, he had worked on E. coli., but in Dr. Edelman's lab he switched to working on mammalian cell and developmental biology. He was supposed to establish a system for in vitro fertilization in mice,  but he did not know very much about early embryology and he had only a very small number of eggs to work with. He grew very frustrated. Then, one and a half years later, Mike Jazwinski joined Edelman's lab, and he decided to work with him instead on studying DNA duplication in yeast. That was another huge leap for him, but it was also his first introduction to yeast cells, which he has worked with ever since.

Finally, he was offered a position as a junior professor in Yasuhiro Anraku's lab at the University of Tokyo and was able to return to Japan

For complete interview, read this 2012 interview conducted by Journal of Cell Biology, where Yoshinori Ohsumi explains his progress within the field of autophagy.

Bacteria*Bioengineering*Storage

A new method of data storage that converts information into DNA sequences allows you to store the contents of an entire computer hard-drive on a gram's worth of E coli and perhaps considerably more than that.

Source:

Nobel Prize for RIBOSOME researchers

Three scientists who showed how the information encoded on strands of DNA is translated into thousands of proteins that make up living matter will share the 2009

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is molecular biological technique used for amplifying (creating copies of) DNA without the use of living organisms such as E coli, yeast. It is used in medical and biological research laboratories for detection of hereditary diseases, diagnosis of infectious diseases, identification of genetic fingerprints, cloning of genes and paternity testing.

The concept of PCR was at first put forward by H Ghobind Khorana et al in 1971 but is seemed to be impractical before gene sequencing and viable thermostable DNA polymerase. Later, after 15 years in 1986 Kary Mills developed the PCR technique. PCR is a process by which DNA is artificially multiplied through repeated cycles of duplication in the presence of DNA polymerase.

The PCR process was patented by Cetus Corporation where Kary Mills worked where he developed the technique. Taq polymerase enzyme was also covered by the patent. The pharmaceutical company Hoffmann-La-Roche purchased the right to patent in 1992 and currently holds them.

DNA polymerase occurs naturally in living organisms and functions to create copies of DNA when cell divides. It functions by binding to single stranded DNA and creating complementary strand. The original concept of PCR technique developed by Mills uses the enzyme in vitro. Double stranded DNA was separated into two single strands by heating at 96 degree C. However, at this high temperature, DNA polymerase was destroyed and required to be replenished after heating stage of each cycle. Thus, it required great deal of time, large amount of DNA polymerase and continued attention throughout the PCR process.

Later, this PCR process was modified by using DNA polymerase obtained from thermophilic bacteria that grow in geysers at 110 degree C. This DNA polymerase was thermostable and do not break down when the reaction mixture was heated to separate strands.

The first thermostable DNA polymerase was obtained from Thermus aquaticus and called Taq polymerase. One of the disadvantages of this Taq polymerase was that it sometimes maked mistakes while making copies of DNA leading to mutation of DNA sequences since it lacked 3’-5’ proofreading exonuclease enzyme. The polymerase Pwo and Pfu obtained from Archaea contained exonuclease enzyme and reduced the mistakes while making copies of DNA. The combination of Taq and Pfu is available now a days that provides both fidelity and accurate amplification of DNA.

PCR amplifies short, well defined DNA fragment. It requires a single gene or just a par of gene. As opposed to living organism, PCR can make copies of only short DNA fragment upto 10 kb ie 1000 base pairs. DNA is double stranded and therefore it is measured as complementary DNA building block (nucleotides as base pairs).

PCR requires
DNA template containing the region of DNA fragment to be amplified
Two primers determining the beginning and end of DNA fragment to be amplified
DNA polymerese to make copies of DNA fragment to be amplified
Nucleotide from which DNA polymerase synthesize DNA strand
Buffer for creating optimum chemical environment for DNA polymerase to perform

PCR is carried out in thermal cycler. It is a machine that cools and heats the reactions tubes within it in precise temperature that is required for each step of the reaction. Evaporation of the reaction mixture is prevented by placing heated lid on reaction tube or by placing thin oil layer on the reaction mixture.

Primer
DNA fragment to be amplified is determined by selecting the primer. Primers are artificial, short DNA strands upto 50 nucleotides that exactly match the beginning and end of the DNA strand to be amplified. They anneal with DNA template at these beginning and end points and DNA polymerase binds and begins synthesis of DNA strand.

The choice of the length of the primers and their melting temperature depends on several considerations. Melting temperature of primer- not to be confused with the melting temperature of DNA at firs step of PCR- is the temperature at which half of the primer binding sites would be occupied. Melting temperature increases with the length of the primer. Short primers would anneal at several points on the long DNA template resulting non specific copies. On the other hand, length of the primer is limited by melting temperature at which it melts. High melting temperature above 80 degree C will cause problem since the DNA polymerase is less active at this high temperature. The optimum length of primer is 20 – 40 nucleotide wit melting temperature of 60 – 75 degree C.

PCR has a series of 20-30 cycles and each cycle consists of 3 steps-
1st step- Double stranded DNA is heated at about 94-96 degree C to separate the strands. This step is called denaturation and breaks apart the hydrogen bond that binds together two DNA strands. Prior to the first cycle, DNA is denatured for extended time period in order to ensure that both template DNA and primers are separated into single strand. The time of this step is usually 1-2 minute/s.

2nd step- After denaturation, temperature is lowered so that primer anneals with the single stranded DNA. This step is called annealation. Temperature of this step depends on the primers and is usually 5 degree C below their melting temperature. Wrong temperature at this step causes primer not to bind with DNA template or binding at random. Time of this step is 1-2 minute/s.

3rd step- After annealation, DNA polymerase has to fill the missing strands. DNA polymerase binds at annealed primer and works its way along the DNA fragment. This step is called elongation. Temperature of this step depends on DNA polymerase. However, time of this step depends both on the DNA polymerase itself and the length of DNA fragment to be elongated. Usually by the rule of thumb, the time of this step is 1 minute for every 1,000 bp.

The PCR product is identified by its size using Agarose gel electrophoresis. The sixe of PCR product is determined by comparing it with DNA ladder.

Uses of PCR
Genetic fingerprinting
Detection of hereditary disease
Cloning of genes
Analysis of ancient DNA
Paternity testing
Genotyping of specific mutation
Mutagenesis
Comparison of gene expression

Genetic fingerprinting is a forensic technique to identify a person by comparing his/her DNA with a sample e.g:- urine, semen, saliva, blood, hair from crime scene can be genetically compared to the blood of suspect.
Genetic fingerprint is unique except for identical twins
Genetic relationship can be determined by comparing two or more genetic fingerprints for paternity test
A slight variation of this technique can be used to determine evolutionary relationship between organisms.