Tag: PCR

POLYMERASE CHAIN REACTION: PRINCIPLE AND APPLICATIONS

BY DAKSHITA NAITHANI

The polymerase chain reaction (PCR) is an in-vitro (laboratory) technique used to produce huge amounts of DNA.

•             PCR is a cell-free amplification method that produces billions of identical copies of any DNA of interest. PCR, which was invented by Karry Mullis in 1984, is today regarded as a fundamental technique for molecular methods. It is the most widely used approach for multiplication of target nucleic acids.

•              The method usually combines complementary nucleic acid hybridization and nucleic acid replication principles, which are applied repeatedly over many cycles to amplify a single and original copy of a nucleic acid target, which is often undetectable by standard hybridization methods, and multiply to 107 or more copies in a short amount of time. In result, it gives a large number of targets which may be identified using a variety of ways.

ADVANTAGES:

•             Despite being simple it is a very powerful technique.

 •            It enables for massive amplification of any particular sequence of DNA given that short sequences on each side of it are known.

•             Improves sensitivity and specificity while allowing for speedier diagnosis and recognition.

PRINCIPLE OF PCR:

Double-stranded DNA in question is denatured, resulting in two independent strands and  each strand is allowed to hybridise using a primer (renaturation). The enzyme DNA polymerase is used to synthesise DNA from the primer-template duplex. To create various forms of target DNA, the three processes of denaturation, renaturation, and synthesis are performed numerous times.

ESSENTIAL REQUIREMENTS FOR PCR:

•             A target DNA which is around 100-35,000 bp in length.

•             Two primers (synthetic oligonucleotides of 17-25 nucleotides length ) that are complementary to regions flanking the target DNA.

•             Four deoxyriobonucleotides  are used(d ATP, d CTP, d GTP, d TTP)

•             MgCl2 (Magnesium Chloride)

•             Nuclease free water

•             Taq DNA polymerase buffer

•             A thermo-stable DNA polymerase is one that can tolerate temperatures up to 95 degrees Celsius.

The target DNA, two primers (in excess), a thermo-stable DNA polymerase (Taq DNA polymerase), and four deoxyribonucleotides are all included in the reaction mixture. It is a method that includes a series of cycles for DNA amplification.

KEY FACTORS OPTIMAL FOR PCR:

•             PRIMERS:

When it comes to determining PCR, these are crucial. Primers with no secondary structure and no complementarity amongst themselves (17-30 nucleotides) are excellent. In PCR, complementary primers can combine to produce a primer dimer, which can be amplified. The replication of target DNA is prevented as a result of this action.

•             DNA POLYMERASE:

Because it can resist high temperatures, Taq DNA polymerase is chosen. After the heat denaturation stage of the first cycle, DNA polymerase is introduced in the hot start procedure. This prevents the misaligned primers from extending, which is common at low temperatures.

Verification or proof reading of exonuclease (3′-5′) activity is absent in Taq polymerase, which might lead to mistakes in PCR products. Tma DNA polymerase from Thermotogamaritama and Pfu DNA polymerase from Pyrococcusfuriosus are examples of thermostable DNA polymerases with proof reading activity.

•             TARGET DNA:

In general, the smaller the target DNA sequence, the greater the PCR efficiency. A mplification of DNA fragments up to 10 kb has been documented in recent years. In PCR, the sequence of the target DNA is also crucial. As a result, CC-rich strand sections obstruct PCR.

•             PROMOTERS AND INHIBITORS:

 Addition of Bovine serum albumin (BSA) improve PCR by shielding DNA polymerase, humic acids which are commonly present in ancient samples of target DNA, hinder PCR.

EACH CYCLE HAS THREE STAGES:

1.            DENATURATION:

The DNA is denatured and the two strands split when the temperature is raised to around 95 degree celsius for about one minute.

2.            RENATURATION OR ANNEALING:

The primers base pair with the complementary regions flanking target DNA strands as the temperature of the mixture is gradually lowered to around 55 degree celsius.  Annealing seems to be the term for this procedure. Due to the high concentration of primer, annealing occurs between each DNA strand and the primer rather than between the two strands.

3.            EXTENSION OR SYNTHESIS:

The 3′-hydroxyl end of each primer is where DNA synthesis begins. By connecting the nucleotides that are complementary to DNA strands, the primers are expanded. The PCR synthesis process is quite similar to the leading strand DNA replication process.  The optimal temperature for Taq DNA polymerase is about 75 degree celsius. (For E.Coli DNA Polymerase is used). By increasing the temperature, the process can be halted (about 95 degree celsius).

Each cycle lasts around 3-5 minutes and in most cases, it is performed on computerised equipment. The corresponding sequence of the second primer lies beyond the new DNA strand linked to each primer. Long templates allude to these additional strands, which will be utilised in the second cycle.

The strands are denatured, annealed with primers, and exposed to DNA synthesis in the second cycle of PCR. Long and short templates are produced at the end of the second round.

The original DNA strands, as well as the short and long templates, are the starting materials for the third cycle of PCR. For each cycle, the procedures are used again and again. About a million-fold target DNA is produced by the conclusion of the 32nd cycle of PCR, according to estimates. As double-stranded molecules build, the small templates containing precisely the target DNA increase.

TYPES OF PCR:

1.            Real-time PCR

2.            Quantitative real time PCR (Q-RT PCR)

3.            Reverse Transcriptase PCR (RT-PCR)

4.            Multiplex PCR

5.            Nested PCR

6.            Long-range PCR

7.            Single-cell PCR

8.            Fast-cycling PCR

9.            Methylation-specific PCR (MSP)

10.          Hot start PCR

11.          High-fidelity PCR

12.          In situ PCR

13.          Variable Number of Tandem Repeats (VNTR) PCR

14.          Asymmetric PCR

15.          Repetitive sequence-based PCR

16.          Overlap extension PCR

17.          Assemble PCR

18.          Intersequence-specific PCR(ISSR)

19.          Ligation-mediated PCR

20.          Methylation –specifin PCR

21.          Miniprimer PCR

22.          Solid phase PCR

23.          Touch down PCR, etc

APPLICATIONS OF PCR:

1.            PCR IN CLINICAL DIAGNOSIS:

PCR’s specificity and sensitivity make it ideal for diagnosing a variety of human illnesses. RFLP is not involved in the development of many genetic diseases (restriction fragment length poly-morphism). For all of these problems, PCR is a godsend since it delivers straight DNA information it is accomplished by amplifying DNA from the appropriate area and then analysing the PCR results directly.

o             PRENATAL DIAGNOSIS OF INHERITED DISEASES:

It is used to diagnose hereditary disorders in the womb utilising chorionic villus samples or amniocentesis cells various c onditions such as sickle cell anaemia, p-thalassemia, and phenylketonuria can thus be identified in these specimens using PCR.

o             DIAGNOSIS OF RETROVIRAL INFECTIONS:

                PCR from cDNA is a useful technique for detecting and maintaining retroviral infections, such as HIV.

o             DIAGNOSIS OF BACTERIAL INFECTIONS:

o             PCR is used for the detection of bacterial infections such as tuberculosis which is caused by Mycobacterium tuberculosis.

o             DIAGNOSIS OF CANCERS:

PCR can identify some virally-induced malignancies, such as cervical cancer caused by the human papillomavirus it can also identify malignancies caused by chromosomal translocations (chromosome 14 and 18 in follicular lymphoma) containing known genes.

o             PCR IN SEX DETERMINATION OF EMBROYS:

The sex of human and animal eggs fertilised in vitro may be identified using PCR using sex chromosome-specific primers and DNA probes. This method can also be used to identify sex-related abnormalities in fertilised eggs.

2.            PCR IN DNA SEQUENCING:

 The process is useful for sequencing since it is considerably easier and faster to amplify DNA. Single strands of DNA are required for this function. Asymmetric PCR involves preferred amplification of a single strand. Strand removal can also be accomplished by digesting one strand.

3.            PCR IN FORENSIC MEDICINE:

For amplification, a single molecule from any source (blood strains, hair, semen, etc.) of a person is sufficient. As a result, PCR is critical for crime detection.

4.            PCR IN COMPARISON WITH GENE CLONING:

In comparison to traditional gene cloning procedures, PCR offers a variety of benefits. Improved efficiency, small amounts of beginning material (DNA), cost-effectiveness, low technical expertise, and the time frame are only a few of them. In the long run, PCR may be able to replace most gene cloning applications.

5.            PCR IN GENE MANIPULATION AND EXPRESSION STUDIES:

The benefit of PCR is that the primers do not need to be complementary to the target DNA. As a result, it may alter and amplify the nucleotide sequence in a portion of the gene (target DNA). The coding sequence of a protein of interest can be changed using this approach. Gene manipulations are also crucial for studying the impact of factors on gene expression.

The study of mRNAs, which are the results of gene expression, requires the use of PCR. Reverse transcription-PCR is used to accomplish this.

6.            PCR IN COMPARITIVE STUDIES OF GENOMES:

PCR using random primers can be used to assess the differences in the genomes of two species. Electrophoresis is used for separation of products for their comparative identification and it is predicted that two genomes from closely related species will produce more comparable bands.

The study of evolutionary biology, more especially phylogenetic biology, relies heavily on PCR. It has transformed palaeontology and archaeological research since it can amplify even minute amounts of DNA from any source (hair, mummified tissues, bone, or any fossilised material).

EXTREMOPHILES: SALINITY AND AT LOW NUTRIENT LEVELS

BY DAKSHITA NAITHANI

Prokaryotic life has dominated much of our planet’s evolutionary history, developing to fill nearly every possible environmental niche. Extremophiles are one of these. Extremophiles have been identified on Earth that can survive in conditions that were previously considered to be inhospitable to life. Heat, extremely acidic conditions, extreme pressure, and extreme cold are examples of extreme environments. The thermophiles were the first extremophiles to be discovered in the 1960s by Thomas Brock of Indiana University. He was investigating life in Yellowstone National Park’s super-hot water pools. He discovered tiny microorganism mats at Octopus Spring in 1965, when temperatures reached 175 degrees Fahrenheit. Thermus aquaticus was discovered, which led to the discovery of PCR and the creation of a new multibillion-dollar enterprise.

EXTREMOPHILES IN SALINITY: HALOPHILES

The halophiles live in high salt concentrations and are named after the Greek term for “salt-loving.” While the majority of halophiles belong to the archaea domain, some bacterial halophiles and eukaryotic species, such as the alga Dunaliella salina and the fungus Wallemia ichthyophaga, do not. Carotenoid chemicals give certain well-known species, such as bacteriorhodopsin, a red hue. They may be found in salty water bodies such as the Great Salt Lake in Utah, Owens Lake in California, the Dead Sea, and evaporation ponds, where the salt content is more than five times that of the ocean. They’re thought to be a viable contender for extremophiles living in Jupiter’s Europa and other comparable moons’ salty subsurface water oceans.

CELLULAR ADAPTATIONS BY HALOPHILES

High salt-in strategy

The high-salt-in approach protects halophiles from a saline environment by accumulating inorganic ions intracellularly and balancing the salt content in their surroundings through KCl influx. Cl- pumps, which are only found in halophiles and transfer them from the environment into the cytoplasm, are involved in this process. Extreme halophiles of the archaeal and bacterial families keep their osmotic equilibrium by concentrating K + inside their cells. The membrane-bound proton-pump bacteriorhodopsin works to accomplish this.

Low-salt, organic solute-in strategy

The high-salt-in approach necessitates physical modification of all macromolecules in order to survive in a very saline environment, which is incompatible with the survival of moderate halophiles that flourish in salinity-varying environments. Osmolytes protect microbial proteins against dissociation in low-salt water while also improving the bacteria’ tolerance to drastic changes in external saline conditions. Glycine betaine was the first bacterial osmolyte discovered in Halorhodospria halochloris.

The majority of halophiles are unable to thrive outside of their high-salt natural habitats. Many halophiles are so delicate that putting them in distilled water causes them to lyse due to the shift in osmotic circumstances. Halophiles include phototrophic, fermentative, sulfate-reducing, homoacetogenic, and methanogenic species in anaerobic conditions whereas in aerobic conditions include phototrophic, fermentative, sulfate-reducing, homoacetogenic, and methanogenic species.

The Haloarchaea, notably the Halobacteriaceae family, belong to the Archaea domain and make up the bulk of the population in hypersaline settings. The family currently has 15 recognised genera. Bacteria (mostly Salinibacter ruber) can make up to 25% of the prokaryotic community, although it usually makes up a considerably smaller portion of the overall population. In this habitat, the alga Dunaliella salina can sometimes thrive.

EXTREMOPHILES AT LOW NUTRIENT LEVELS: OLIGOTROPHS

An oligotroph is an organism that can survive in a low-nutrient environment. Oligotrophs are usually known for their sluggish development, low metabolic rates, and sparse population density. The settings are ones that provide little in the way of life support. Deep marine sediments, caverns, glacial and polar ice, deep underground soil, aquifers, and leached soils are examples of these habitats.

The cave-dwelling olm the bacteria Pelagibacter ubique, which is the most numerous creature in the seas and lichens with their incredibly low metabolism are all examples of oligotrophic species.

Caulobacter crescentus is an oligotrophic Gram-negative bacteria found in freshwater waterbodies. The whole cell functions as an integrated system in the control circuitry that controls and paces Caulobacter cell cycle development. As it orchestrates activation of cell cycle subsystems and Caulobacter crescentus asymmetric cell division, the control circuitry monitors the environment and the internal status of the cell, including the cell topology. The control system has been meticulously tuned as a whole system for reliable functioning in the face of internal stochastic noise and external unpredictability by evolutionary selection.

The bacterial cell’s control system is organised in a hierarchical manner. The signalling and control subsystem communicates with the outside world through sensory units that are mostly found on the cell surface. To adjust the cell to current conditions, the genetic network logic responds to signals received from the environment as well as internal cell status sensors.

ENVIRONMENT AND LOCATIONS

Oligotrophic lakes are often found in northern Minnesota, with deep clear water, stony or sandy bottoms, and minimal algae.

Oxygen levels are high throughout the water column in oligotrophic lakes. Cold water may store more dissolved oxygen than warm water, thus oligotrophic lakes’ deep regions remain quite cold. Low algal content also provides for more light penetration and less breakdown. Algae, zooplankton, and fish die and are degraded by bacteria and invertebrates at the bottom of the ocean. The process of breakdown consumes oxygen. 

Locations

 Oligotrophs and eutrophs coexist in natural ecosystems, and their proportions are determined by an individual’s capacity to prevail in a given environment.  Despite their capacity to exist in low-nutrient settings, they may struggle to survive in nutritionally- rich ones. Most microorganisms are not well adapted to exist in nutrient-limited circumstances and frigid temperatures (below 5 °C), Antarctic habitats offer very little to sustain life. Some of the documented examples of oligotrophic environments in Antarctica are:

Lake Vostok, a freshwater lake cut off from the rest of the world by 4 kilometres (2.5 miles) of Antarctic ice, is often cited as a prime example of an oligotrophic ecosystem. Because of the lake’s severe oligotrophy, some people assume that sections of it are entirely sterile. This may be used as a model to simulate alien life investigations on frozen planets and other celestial worlds.

Oligotrophic soil environments

In general, nutrient availability decreases as the depth of the soil environment increases, since organic molecules degraded from detritus are swiftly eaten by other microorganisms on the surface, resulting in nutritional deficiency in the deeper levels of soil.

Collimonas is one of those species that may survive in an oligotrophic environment as it has the capacity to not only hydrolyze the chitin generated by fungus for nutrition, but also to create materials. Fungi are a prevalent element of the habitats where Collimonas thrives. In oligotrophic settings, reciprocal relationships are prevalent. Weathering also allows Collimonas to access electron sources from rocks and minerals.

The environment of soil in polar locations, such as the Antarctic and Arctic regions, is termed oligotrophic since the soil is frozen and biological activity is minimal. Actinobacteria, Proteobacteria, and Cyanobacteria are the most common bacteria in frozen soil, with a tiny quantity of archaea and fungus. Under a wide range of low temperatures, actinobacteria can keep their metabolic enzymes active and continue their biochemical processes.

The following are the characteristics that a bacterium should have in order to be labelled as an oligotroph:

(a) Having a form with a high surface-to-volume ratio.

(b) Having an innate propensity for using metabolic energy for food absorption during phases of growth stagnation.

(c) Possessing nutrition absorption abilities that are expressed in a constitutive manner.

(d) Presence of a low-specificity, high-affinity transport mechanism that allows for simultaneous absorption of mixed substrate.

 (e) Having systems for conserving nutrition after it has been absorbed.

Extremophiles and their products have revolutionised many aspects of our home and professional life, from household materials to molecular diagnostics. It is not unlikely that new and medically useful discoveries will be found in the realm of extremophile research; the potential of these organisms is so fresh and huge that their applications may be restricted only by imaginations.