Top 10 Model Organisms

Conducting trials directly on humans is forbidden on humanitarian grounds. Since the human body is very complex direct involvement of humans as subjects of experiments may not always be possible. Therefore it is ideal to use prokaryotes, eukaryotes, and organisms other than humans. These helper organisms are known as model organisms. They contribute to scientific and industrial research. These organisms include bacteria, fungi, viruses, algae, nematodes, worms, and higher animals. Model organisms are selected based on criteria such as their ease of growing, short life cycle, well-defined cellular structures, easily analyzable properties, safe and ethically sound. Using hazardous toxin-producing bacteria or organisms for conducting experiments is forbidden and is accessed only in rare and unavoidable cases. Only trained, well-qualified professionals handle these organisms. Model organisms are involved in genome analysis, recombination experiments and sequence similarity studies. It requires a lot of care and caution to handle these organisms. Recombinant vaccines, peptides, and biomolecules are produced using model organisms. Biosatellites and spaceships use model organisms in their first launch.

1.   Yeast:

Yeast is a very useful eukaryote with beneficial properties. Yeast is very easy to culture because it can easily grow on fruits, sugary and salty foods. It can easily proliferate from 12⁰C to 40⁰C. The environment of acidic to alkaline is suitable for its growth. Beer and wine industry widely make use of yeast. Preparation of bakery products also involves yeast. Division of yeast occurs due to a process is known as budding. Yeast cells are easy to study since yeast is a single-celled organism. In addition to studying yeast cells with ease, these cells are comparatively safe to handle.

Most of the experiments in genetic engineering and genome research involve the use of yeast as a model organism. It is possible to study cell cycle, DNA replication, protein and cell wall synthesis using yeast cells. Gene cloning can be achieved using vectors. Yeast artificial chromosomes (YACs) are yeast cloning vectors. Typical features of YACs include telomere (TEL), centromere (CEN), selectable markers on each arm, an origin of replication, and restriction sites or multiple cloning sites. YAC vectors can accommodate DNA fragments of several base pairs. Protein-protein interaction studies make use of the yeast two-hybrid system. Cancer genetic studies also involve yeast as model organisms.

2.   Fruit fly:

Most of the genetics experiments involve the use of the fruit fly. It is scientifically known as Drosophila melanogaster. Drosophila is easy to grow since it does not require any specific conditions to grow. It can grow on a wide range of foodstuffs right from a piece of banana to a culture medium. There is a wide range of mutants observed in the fruit fly. Studying its genes is simpler as compared to other organisms. Drosophila is used to study developmental genes, mutant genes, and chromosomes. Morgan was the first scientist to use this model organism. The generation time of fruit fly is rapid as it takes less than two weeks to grow. Transgenic fruit flies are those that consist of foreign gene(s) inserted into their genome.

Drosophila is one such genus of the fruit flies that contains over 900 described species. The genic balance theory was used to identify the concept of sex determination. This theory worked out primarily in Drosophila. According to this theory, sex determination is the ratio of sex chromosomes and polytene chromosomes. A polytene chromosome is a giant chromosome in Drosophila produced by the endomitotic process. It consists of many chromatids. DNA replication and heterochromatin studies are carried out using Drosophila polytene chromosomes. They are also involved in gene mapping and linkage studies. The fruit fly is a model system for understanding human biology and disease. Findings suggest that this model organism has homologs for 177 out of 289 genes involved in human cancers.

Image: Model organisms

3.   E.coli:

Escherichia coli are most widely used bacteria in the biotechnology industry. Genetic engineering principles make very high use of this bacteria for cloning and other experiments. The life cycle of E. coli is simple to understand. Hence they are easy to grow and safe to handle. These bacteria consist of extrachromosomal material known as plasmids. Genetic modifications are possible with this model organism. E.coli can be engineered to produce the desired product. For example, gene modification helps in synthesizing insulin. Plasmid cloning vectors include pUC 19 vectors that contain multiple cloning sites. Important applications in model organisms are as follows:

a.    Gene cloning experiments: Pure samples of cloned DNA are obtained using plasmid vectors. Some of the plasmids with multiple cloning sites enable different types of restriction enzymes to act on them.

b.   Bacteriophage studies: The viruses that infect the bacteria are known as bacteriophages. They follow lytic as well as the lysogenic cycle of infection.

c.     Gene mapping: E.coli bacteria undergo a process of conjugation for transferring genes from one bacterium to another. Thus, gene mapping is possible with the help of conjugation experiments.

d.    Study of DNA replication: Prokaryotic DNA replication study involves E. coli as a model organism.

e.     Identification of mutant phages: E. coli are used to identify mutant phages in site-directed mutagenesis.

f.       Bacterial Artificial Chromosomes (BACs): They are useful for cloning DNA fragments in E. coli. BACs consists of an origin of replication, multiple cloning sites, a selectable marker, and other features.

4.   Nematode C. elegans:

Caenorhabditis elegans is the widely used nematode in zoology. This nematode exhibits anatomical simplicity. It is ideal to carry out gene diversity and cell cycle studies with this organism. The life cycle of this nematode is just three days. Early embryonic genes are clear to study with this organism. It is capable of learning simple tasks. C elegans is used to study the genetic and molecular aspects of embryonic development, morphogenesis, nerve systems, aging, and behavior.

5.   Arabidopsis thaliana:

It is a small plant popular for studying genetic analysis. A complete genome sequence of A. thaliana is available. It is useful to understand processes including nutrient transport and flower development. CRISPR/Cas 9 gene editing also utilizes this plant.

6.   Mus musculus:

The gene content in mice is similar to that of humans. The genome sequence of mice is known to us. Mouse models are used to study organ and immune systems. Mice are just like humans in developing diseases such as cancer, diabetes, atherosclerosis, hypertension and Alzheimer disease. Thus mouse models are far better to work. The mice help in studying various gene mutations, biochemical pathways, metabolism, pharmacokinetics, and pharmacodynamics. Thus, they are ideal for clinical trials and research.

7.   Neurospora crassa:

It is a haploid fungus widely used in cellular processes. It is possible to grow this strain and use it for tetrad analysis. Typical findings using this model organism involve centromere distance, crossing over and poky mutants. The recessive traits easily show up in the offspring. Thus it is simple to study genetic analysis.

8.   Danio rerio:

The common name for Danio rerio is zebrafish. It is a model organism in stem cell biology and developmental genetics. Its embryos are transparent and help in clearly identifying stages of development. It is easy to feed zebrafish by making a genetic cross.

9.   Lambda bacteriophage:

It is a phage that infects E. coli cells. It replicates using bacterial machinery. A bacteriophage is an important tool for genetic analysis. It is a model system for studying genetic recombination, complementation, and cloning experiments. The lambda genome is capable of insertion into the bacterial chromosome.

10.                      Cavia porcellus:

The common name for this model organism is the guinea pig. However, this organism is not a pig but a rodent. Germ theory was established using guinea pigs as model organisms. They are used to study infectious diseases such as cholera, typhus, and brucellosis.

References:

[1] Biotechnology, David P. Clark, Nanette J. Pazdernik

[2] Introduction to Genetic Analysis, Anthony J.F. Griffiths, Susan R. Wessler, Richard C. Lewontin, Sean B. Carroll

[3] Genetic Analysis: Genes, Genomes, and Networks in Eukaryotes, Philip Mark Meneely

[4] Encyclopedia of Genetics, Eric C.R. Reeve

[5] Neurospora: Contributions of a Model Organism, Rowland H. Davis
[6] Model organisms- Wikipedia

© Copyright, 2018  All Rights Reserved

Northern blotting technique

Electrophoretically separated RNA molecules get transferred from the gel to the absorbent sheet, immersed in a labeled probe for hybridization. Thus, the northern blotting follows the same steps of Southern blotting. However, RNA instead of DNA gets blotted. The standard Northern blotting procedures help to compare the quantities of the transcripts from different tissues. The technique’s sensitivity increases to a 100 fold using an mRNA or a messenger RNA. The technique analyses many types of RNAs such as micro RNA, small nuclear RNAs or snRNAs, small interfering RNAs or siRNAs, and mRNA. The technique is helpful in RNA interference studies or RNAi technology. It also involves studying the expression of oncogenes. Northern blotting includes human RNAs, plant, and animal RNAs. The technique has placed itself very well in genetic research and engineering.

Image: Northern blotting

The description of the blotting procedure is as follows:

Step 1: Isolation of mRNAs:

RNA extraction uses a homogenized tissue sample. Oligo (dT) cellulose chromatography helps in isolating mRNA with a poly (A) tail. We know the mechanism of polyadenylation well. It protects the RNA from degradation by adding polyadenine nucleotides to the RNA molecule. Widely used methods include phenol-chloroform extraction or trizol method.

Step 2: Electrophoresis:

Once the RNA isolation gets completed, the loading of the samples in the wells helps to obtain the bands. Separation of the RNA molecules involves agarose gels most of the times. Fragmented RNA or micro RNA separation may include polyacrylamide gels. Formaldehyde acts as a denaturing agent in the electrophoresis. It limits the RNA secondary structure. Fragment sizes comparison uses a ladder RNA in another well. Electrophoresis involves staining the gel with ethidium bromide. Handling ethidium bromide requires caution because of its carcinogenicity and neurotoxicity. Ethidium bromide intercalates the RNA, thereby acting as an intercalating agent.

Step 3: Using a nylon membrane:

After separation of RNA molecules through gel electrophoresis, the samples get transferred to a nylon membrane through a capillary system. Following is the apparatus for northern blotting:

The first step involves filling a tray with a buffer solution consisting of formamide. It helps in lowering the annealing temperature of the probe-RNA interaction thereby preventing RNA from getting degraded. Then the RNA gets immobilized to the membrane through a covalent linkage. UV light or heat facilitates this property. The blotting paper used in this apparatus plays a role in capillary action by carrying the buffer solution through the gel. Once the RNA interacts with the buffer, it gets transferred to the membrane. A stack of paper towels and weight kept on the membrane enhances the imprint of the bands on the membrane. Now the RNA molecules present on the membrane exactly resemble as they were on the gel.

Step 4: Hybridization with the labeled probe:

Exposure of the membrane to the probe ensures hybridization of RNA molecules. Northern blotting procedure involves probes composed of complementary sequences of RNA. It consists of at least 25 complementary bases. Northern blotting uses cDNA as a probe. Radioactive labeling of the probe includes a radioactive isotope P³². Alternative labeling involves non-radioactive techniques such as chemiluminescence labeling. The chemiluminescence technology involves the breakdown of the chemiluminescence substrates through enzymes such as alkaline phosphatase or horseradish peroxidase. The substrates produce a detectable emission of light.

Step 5: The procedure of chemiluminescent labeling:

The probe (cDNA) gets attached to the enzyme alkaline phosphatase or horseradish peroxidase. Alternatively, labeling of the probe may involve a ligand. The ligand gets attached to the enzyme. Determination of the efficiency of the hybridization includes ionic strength, viscosity, duplex length, and base composition. Non-hybridized probes get removed by washing the membrane filter gently.

Step 6: Exposure to the X-ray film:

An X-ray film detects the signals generated with the chemiluminescent-labeled probes hybridized to RNA. A quick and sensitive signal generation occurs through chemiluminescent labels.

Step 7: Quantification of RNA through densitometry:

The process of densitometry involves a quantitative measurement of the optical density in the light-sensitive material.

Following is the summary of the northern blotting procedure:

1.     Isolation of RNA from a tissue sample.
2.     Loading the samples and the markers in the electrophoretic wells.

3.     Fragments get separated based on their sizes. Molecules with larger sizes are near the wells. Molecules with smaller sizes move faster. The fragments appear in the form of bands.

4.     Exposure of the gel to a buffer solution and a membrane filter.

5.     RNA gets transferred from the gel to the membrane.

6.     RNA hybridization using probes.

7.     Signal detection

8.     RNA quantification

Applications of northern blotting:

Gene expression study includes the pattern of gene expression in the tissues, organs and developmental stages. Northern blotting applies in studying the overexpression of oncogenes, the upregulation or downregulation of oncogenes and tumor-suppressor genes. It may help to find a gene function. Northern blotting helps to check the cloned DNA. Especially it checks the cloned cDNA since the technique uses cDNA as a probe to detect specific RNA. Analysis of micro RNAs becomes easy with this technique. A high resolution northern blotting monitors RNA expression. Micro RNAs are short and non-coding regulatory molecules. Post-transcriptional regulation of genes involves the role of micro RNA. The technique is readily available. Hence it is used in micro RNA analysis. The probes used in the technique are known as locked nucleic acid modified oligonucleotide probes. They are extremely sensitive and specific in detecting mature micro RNAs.

Immuno-northern blotting detects RNA modification through antibodies. The RNA gets separated through electrophoresis and transferred to the membrane. Immuno-blotting involves antibodies. It reveals antibody cross-reactions, characterization of antibodies, and modified nucleosides. It is a highly specific technique. The northern blotting technique also characterizes the RNA interference reagents. The comparison of the data derived from deep sequencing of micro RNAs with endogenous and exogenous RNAs is possible with the northern blotting technique. A gene silencing phenomenon is known as RNAi or RNA interference. The double-stranded RNAs get processed into small interfering RNAs (siRNA). A siRNA acts like a guide and enables cleavage of a homologous RNA. It occurs mainly in the RNA induced silencing complex (RISK). High-resolution northern blotting efficiently detects the length heterogeneity of the RNAi technology reagents.

A blot-base is an online database used to publish northern blots. It is a database used in genome sequencing, determination, and the protein structure.

Reverse northern blotting:

It is a variant of northern blotting technique. In this, the DNA fragments get hybridized with the RNA probes labeled radioactively. It enables gene expression profiling.

Virtual northern blotting:

It involves a comparison of relative amounts of transcripts in different tissues. The comparison is between small quantities of total RNA and full-length cDNA.

Advantages and disadvantages of northern blotting:

Detection of RNA size and observation of alternate spliced products become easy with this method. The technique is applicable for quantitative as well as qualitative analysis. It has a high specificity. With so many advantages, there are disadvantages too. As compared with RT-PCR, northern blotting has a low sensitivity. Analyzing thousands of genes at a time is not possible. The RNases are always ready to degrade the sample. Hence the technique always requires RNase inhibitors and proper sterilization of glassware. Chemicals used in northern blotting may be risky.

References:
[1] Molecular Biology Techniques: An Intensive Laboratory Course, Walt Ream, Katharine G. Field

[2] Ana Techniques in Biotechnology, Goutam Bhowmik

[3] Gene Cloning and DNA Analysis, T.A. Brown
[4] Molecular Plant Biology: A Practical Approach, Volume 2, Philip M. Gilmartin, P. M. Gilmartin, Dr. Chris Bowler

                                  © Copyright, 2018 All Rights Reserved.

Southern blotting technique

The gel electrophoresis technique separates the DNA fragments based on their sizes. Smaller molecules move faster than the larger molecules. A basic question arises as to why separate these fragments? The reason is very simple. Separation of fragments based on the sizes helps to obtain specific fragments from the gel instead of getting the entire genomic DNA. Hybridization involves finding the location of a gene or its product using a nucleic acid probe. Most of the times, the probes are small single-stranded DNA molecules. Determination of complementary sequences utilizes hybridization techniques. Hence, the probes bind only to the complementary sequences. Isolated bands from electrophoretic technique determine an efficient mapping of DNA sequences or gene detection. Blotting technique facilitates hybridization. The process involves the transfer of bands to a nitrocellulose membrane. There are three types of blotting procedures depending on the type of the molecule. Southern blotting is used to blot the DNA. Northern blotting is used to blot the RNA. Western blot involves the transfer of protein bands. E.M. Southern derived the southern blotting method for the first time.

Image: Southern blotting

Analyzing the sequences using southern blotting:

Step 1: Treatment with a restriction enzyme:

The DNA undergoes a treatment with a restriction enzyme. The enzymes cleave the DNA into various fragments. The process of cleaving the DNA to obtain fragments is known as restriction digestion. The fragments obtained from restriction digestion are known as restriction digests. Restriction enzymes are known as molecular scissors as they cut the DNA at specific sites known as the restriction sites.

Step 2: Separation of the fragments through gel electrophoresis:

The main aim of restriction digestion involves studying the DNA in bits and pieces and picking up the piece of interest for analyzing. Electrophoresis does the work of separating the fragments as per the sizes. Not only DNA but also RNA can be separated. The principle of electrophoresis is simple. The DNA is a negatively charged molecule. It migrates toward the positive electrode. A positively charged molecule moves toward the negative electrode. The shape of the molecule, the charge, and the molecular length determine the rate of migration. Only one criterion of gel electrophoresis involves molecular length. The composition of the gel mainly constitutes agarose, which is nothing but a network of pores through which DNA molecules travel. Molecules of different lengths form bands on the gel.

Step 3: Staining the DNA

Staining the DNA involves ethidium bromide. This chemical is a carcinogen and neurotoxic. Use it with precaution. Staining with the ethidium bromide helps in visualizing the bands under ultraviolet light. Ethidium bromide intercalates with the DNA.

Step 4: Transferring the gel to a membrane filter:

The gel consisting of DNA fragments gets transferred to the membrane filter. Following description is about the apparatus. Firstly, a buffer solution poured into a tray serves as an alkaline medium. Soaking the gel in the buffer solution denatures the DNA into single strands. Next step involves neutralization of the gel and placing the blotting paper. The ends of the paper act as a wick that takes up the buffer solution until the gel. The membrane filter covers the gel. The next step involves placing the paper towels and weight on the filter. Due to the blotting action, the buffer solution travels through the gel onto the membrane filter. The DNA fragments get picked up by the buffer solution and get transferred to the membrane.

Step 5: Hybridization with the probes:

The probes may or may not be radioactively labeled. The process involves the addition of the probe to the membrane filter so that the DNA present on the filter gets hybridized with the probe.

Step 6: Autoradiography:

Permanent fixation of the DNA on the membrane involves heating at 80⁰C for 2-3 hours. Now, the DNA gets completely hybridized with a labeled DNA probe. The probe forms a complementary base pair with the homologous sequence on the DNA fragment. Unbound probes are removed by carefully washing the membrane. Autoradiography technique involves an X-ray sensitive photographic film. Exposing the membrane filter to the X-ray sensitive photographic film determines the labeled molecules.

In summary, the southern blotting technique involves restriction digestion, gel electrophoresis, probing and autoradiography.

Applications of the southern blotting:

1.     SNP analysis:

Single base pair changes constitute single nucleotide polymorphisms. Southern blotting efficiently determines SNP alleles. The initial step involves the isolation of genomic DNA and digestion with the restriction enzymes. The electrophoretic techniques separate the fragments based on their sizes in kilobases. The action of the blotting paper helps in transferring the DNA present on the gel to the membrane filter placed on top of the gel. A stack of paper towels and weight kept above the membrane helps in keeping the membrane fixed at one place. Hybridization with the probe enables complementary base pairing. The visualization of the bands under an X-ray sensitive photographic film gives a clear picture of the DNA. Southern blotting helps in detecting homozygotes and heterozygotes. Comparison of bands becomes easy with the southern blotting.

2.     DNA molecular testing with ASOs:

It includes allele-specific oligonucleotide hybridization or short oligonucleotides complementary to SNP alleles. The oligonucleotides mixed with DNA get hybridized. ASO hybridization also involves Southern blotting. The ASOs labeled radioactively get hybridized with the DNA immobilized on the membrane filter. Analysis of the resulting autoradiograms helps in detecting gene mutations.

3.     RFLP analysis:

RFLP analysis includes detection of genetic disorders such as PKU, sickle cell anemia, and many others. Restriction fragment length polymorphisms or RFLP analysis exploits homologous DNA variations.

4.     Zoo blot:

A blot consisting of DNA from a variety of organisms is known as a zoo blot. The digestion of DNA obtained from organisms such as chicken or a hamster with the restriction enzymes gives fragments of different lengths. The analysis of these fragments includes southern blotting.

5.     DNA typing or DNA fingerprinting:

Digestion of DNA with endonucleases giving fragments, later on electrophoresed, give banding patterns on the gel. The southern blot of the probe gets further probed with the VNTR-specific probes in the DNA fingerprinting technique. Applications of DNA fingerprinting include paternity and maternity testing, studying mitochondrial inheritance, and crime scene investigation.

6.     DNA microarray involving southern blotting:

Southern hybridization with DNA microarray includes unlabelled DNA probes targeting label-free DNA molecules. However, DNA microarray uses a chip or a probe array. The method uses fluorescence dyes or cyanine dyes.

References:
[1] Molecular Biology Techniques: An Intensive Laboratory Course, Walt Ream, Katharine G. Field

[2] Ana Techniques in Biotechnology, Goutam Bhowmik
[3] Gene Cloning and DNA Analysis, T.A. Brown

                                       © Copyright, 2018 All Rights Reserved.

Emergence of PCR technology in the biotech industry

The biotech sector involves a set of methods including the drugs, biologics, devices and other applications by integrating natural sciences and engineering principles. These biotech firms generally compete in established markets with novel products and technologies. Lab-based genome replication was tough to achieve since the techniques were inefficient and crude before the advent of PCR technology. However, the biotech industry started emerging in the late 1980s after the invention and approval of PCR technology. Since most of the products require replication or amplification of the genetic sequences, PCR helped to establish the biotech market. Hence, it has been a breakthrough in the world of life sciences. The simple initial design of the PCR technique had various improved versions later on with automated functioning. It not only improved the CAGR of the biotech industry but also helped the other fields to develop including the Human Genome Project, forensics, biodiversity, animal technology, and agriculture.

Image 1: Polymerase Chain Reaction

A biotech firm known as Cetus Corporation in the early 1970s was the first company to bring forward the concept of the PCR. A scientist working at Cetus Corporation known as Kary Mullis worked hard to achieve the DNA amplification using various molecular biology techniques. He first tried synthesizing oligonucleotide molecules manually and then tried evaluating them through automated sensitizer prototypes. Cloning was prevalent before PCR technology. However, PCR helped in improvising the technique. Cloning DNA was a time-consuming process. Thus, scientists tried to find a technique which would speed up the process generating more number of gene copies. PCR manifested this concept practically. The PCR technology was successful in the 1980s. Earlier the cloning techniques were “in vivo” cell-based. Now, most of the techniques involve in vitro polymerase chain reaction.

About Polymerase Chain Reaction (PCR):

In a process known as amplification, PCR produces millions of copies of short DNA segments through repeated cycles of denaturation, annealing, and extension. Thus, it generates a huge quantity of DNA to perform various tests. The PCR does not require the host organism. The amplified PCR products are known as amplified DNA or amplifiers. PCR is a complement to the cloning. However, it is not a complete replacement. PCR involves a principal of thermal cycling. The exposure of the reactants to repeated heating and cooling cycles is temperature dependent. It is a DNA melting and enzyme-based quick type of replication involving a selective amplification. Once a DNA gets amplified, it serves as a template in carrying out a series of reactions. It leads to a very high rate of DNA amplification. A PCR machine or a thermal cycler possesses a capacity to produce huge quantities of DNA fragments if the base pair sequence of the fragment is known. The PCR machine needs a very small quantity of the sample. It produces millions of copies of fragments.

Image 2: Steps involved in Polymerase chain reaction

Steps involved in PCR:

Three main steps of PCR include denaturation, annealing, and extension.

1.     Denaturation: The DNA double helix requires unwinding to undergo replication. Hence, the denaturation process primarily denatures the DNA double helix into a single-stranded structure. Denaturation process requires a temperature of 90-95⁰C. At this temperature, the hydrogen bonds break apart.

2.     Annealing: The denatured solution is cooled to carry out the process of annealing. The primers anneal at 37-65⁰C. The primers are complementary DNA sequences. They involve annealing to the opposite strands of the template near the desired sequences. Thus, hybrid DNA molecules paired with strands get synthesized. Annealing leads to spontaneous alignment of the two complementary strands to form a double helix.

3.     Extension: An enzyme known as Taq DNA polymerase extends the primers. The enzyme requires a temperature of 70-75⁰C. We get the enzyme from thermophilic bacteria known as Thermus aquaticus.

Repetition of the heating and cooling cycles is a process involved in thermal cycling. Repeated thermal cycling results in an increase in the amount of unit-length DNA geometrically. Various thermal cyclers used in the industry include commercial applications for research purpose. However, there are limitations and strengths of this technology. Difficulty in the purification of gene fragments, cumbersome clone identification, and unattainable fragments with more than 100 Kb sizes are few limitations of the technology.

Applications of PCR:

The primary strength of PCR includes products easily obtained from a wide number of DNA samples. PCR is used to screen the DNA mutations, find SNPs and variations in the lengths of the microsatellites. PCR amplification requires a very small sample. A blood spot or a piece of hair is enough for the test. Hence, PCR has wide applications in the fields of forensics and archaeology. In clinical diagnostics, PCR involves detection of virus-induced diseases such as HIV, and virus-induced cancer. Thus it helps in treating the diseases. With the help of amplified samples, it becomes easy to run the gel and carry out the southern blot to reveal paternal and maternal relationships for conducting a successful DNA fingerprinting test.

With PCR technology, detection of pathogens, mutation screening, and genetic matching is easy. Human Genome Project achieved great heights due to PCR. It involves sequencing and bioinformatics apart from gene cloning. Site-directed mutagenesis and gene expression studies in genetic engineering require PCR. Plant genetic research essentially involves PCR.

PCR Market data:

The global industrial analysis and market opportunity studies reveal the expansion of the PCR market at a considerable CAGR. Hence there is a huge demand for PCR machines, reagents, kits, and other PCR consumables. Instruments including standard PCR systems, digital and RT-PCR systems have a great demand. The machine has high precision accuracy, reproducibility, and speed. Biotech companies in the Asia Pacific and North America expand in the PCR technology. More and more pathology labs have started using PCR for the detection of various diseases.

FDA approval for PCR:

Multiplex PCR, RT-PCR, and many other types of PCR gained approvals from the Food and Drug Administration, FDA every year. These assays further involve detection of HIV, HCV, HBV, Mycoplasma, and other infectious agents. FDA approved nucleic acid testing Cobas 6800 and 8800 PCR systems for studying the zika virus.

Digital PCR has opened up new avenues known as Bridged Nucleic Acid (BNA) technology. A bridged nucleic acid is a six-membered bridged structure. It consists of an N-O linkage. It is a nucleic acid analog with a higher binding affinity, single mismatch discrimination, and many other characteristics. BNA hybrids help to conduct sequence-specific hybridization and specific designing of probes for BNA.

RT-PCR or reverse transcriptase PCR involves a highly sensitive technique for detecting and quantifying RNA. Following steps are involved in RT-PCR:

A cDNA gets synthesized from an mRNA molecule using a primer and a reverse transcriptase enzyme. The cDNA is amplified using PCR. It is used for testing and quantifying RNA.

PCR also helps in plant genome analysis, editing and study of targeted mutagenesis.

References:
[1] Human Molecular Genetics 3, Volume 3, T. Strachan, Andrew P. Read
[2] Understanding PCR: A Practical Bench-Top Guide, Sarah Maddocks, Rowena Jenkins
[3] PCR Market data, Google news
[4] The Polymerase Chain Reaction, Kary B. Mullis, Francois Ferre, Richard A. Gibbs

© Copyright, 2018 All Rights Reserved.

DNA sequencing

The DNA structure consists of four main nucleotide bases such as adenine, guanine, thymine, and cytosine. These nucleotide bases get arranged in a specific order. Thus, it becomes important to determine the order of the nucleotide bases. Recombinant DNA technology involves amplification of a gene using PCR or cloning techniques. The next step involves the determination of the sequence using a DNA sequencing technique. Determining the sequence helps to find out the underlying mutations associated with a specific genotype. It becomes easy to map the human genome and study diseases associated with gene sequence alterations using DNA sequencing techniques. DNA sequencing not only helps in determining the genetic cause of the disease but also aids in the personalized medicine and pharmacogenomics. Although pharmacogenomics relatively new field, it tremendously helps in determining the response of an individual’s genome towards a particular treatment. It becomes easy to locate the restriction sites using DNA sequencing technique. Gilbert and Sanger shared the Nobel prize for finding out this technique. Maxam Gilbert found out chemical sequencing method. Sanger found out an advanced one. The most commonly used method is known as Sanger’s dideoxy method of DNA sequencing.

Image: DNA sequencing

This method helps in sequencing the linear as well as the circular DNA. The first step involves heat treatment for denaturing the DNA into the single-stranded structure. Next step involves annealing the primer to one of the strands. The primer used in the DNA synthesis consists of an oligonucleotide. The DNA synthesized through this step ensures complementarity with the sequence of interest. Each sequencing experiment requires four set of reactions. The components include single-stranded DNA (to be sequenced), oligonucleotide primer, DNA polymerase, nucleotide precursors such as dATP, dCTP, dGTP, dTTP, and a small amount of dideoxynucleotide ddNTP. A dideoxynucleotide is a modified version of deoxynucleotide. Its deoxyribose sugar consists of a 3’-H instead of 3’-OH. Labeling the primers or the precursors helps in determining the newly synthesized DNA. The reactions consisting of the modified deoxynucleotides or the dideoxynucleotides differ from the others. These modified precursors get added in the reaction mixture to about one-hundredth the amount of unmodified precursor. Upon extension of the primer, the DNA polymerase occasionally adds a dideoxynucleotide. After this step, the DNA synthesis stops. Since the dideoxynucleotide does not have a 3’-OH, a new phosphodiester bond cannot form. Hence, every DNA synthesized using this method consists of a dideoxynucleotide at its end.

The gel electrophoresis helps in separating the DNA chains. The DNA bands get revealed using autoradiography. Reading the sequencing ladder from the bottom to the top helps in determining the sequence of the newly synthesized strand.

DNA sequence analysis:

After determining the sequence, they get an entry into the computer databases using sophisticated techniques. The computer programs help in analyzing the restriction sites, homologous sequences, regulatory sequences, and many more. The designing of the computer programs is such that the possible protein coding regions get detected easily. These programs look for the initiator codon in frame with a stop codon. Hence, these programs detect open reading frame. Genes coding a particular protein get identified easily using the DNA sequencing technique. Segments of DNA capable of expressing a particular phenotype also get identified. It is also possible to identify the genes adjacent to the CpG islands, thereby helpful in assessing the disease phenotypes.

Next-Gen Sequencing:

It involves amplification of the cloned segments and rapid sequencing. This process helps in identifying the bases while incorporating them in the nucleotide chain. Every base emits a fluorescent signal thereby simplifying the identification process. It is a highly scalable technology. It gets classified under the high throughput sequencing and involves massively parallel processing. Next-gen sequencing technique helps in sequencing large stretches of the DNA using parallel processing. It also provides a high resolution and specified the view of the bases, gene or the exome. The intensity of the signal helps in measuring the things accurately. Single nucleotide polymorphisms, insertions, deletions, CNVs, and chromosomal aberrations get detected through next-gen sequencing.

Whole genome sequencing:

The entire genome gets analyzed with the help of whole-genome sequencing. The identification of the genes associated with the inherited disorders, cancers, and other conditions becomes relatively easier using whole genome sequencing. Provides a high resolution of the genome and includes variants. Population studies involve the sequencing of large genomes of the animals, plants, and other organisms. Whole genome sequencing works well with the small as well as large genomes.

Other applications of DNA sequencing:

Thousands of diseases follow the Mendelian pattern of inheritance. Identification of the alleles associated with the disease phenotype becomes easy with the help of sequencing techniques. Certain conditions become so complicated that the patient needs the advice of advanced therapeutics such as bone marrow transplantation. The DNA sequencing reports help in decision making and advice. Pharmacogenomics and personalized medicines, though relatively rare fields, prove beneficial for the diagnosis and early treatment of the individual in a highly specific manner. These techniques also depend on the DNA sequences and other intricate details in the genome. Based on the genome constitution of a patient, prescription of a suitable drug becomes easy.

The Human Genome Project got established successfully through sequencing techniques. Without the sequencing techniques, determining the sequences of the large genomes becomes difficult. High throughput sequencing helps in a better understanding of the oncogenes and tumor-inducing genes in the individuals. Scientists worldwide are trying to deal with these genes to study the effects of various environmental factors on the cancer-causing genes. Thus, sequencing plays a very important role in the diagnostics as well as research. The study of the variations in the genetic compositions of the organisms gets a sophisticated approach using high throughput sequencing. Switching on and off the genes also determines cellular processes. They also involve mutations for the same identified through high throughput sequencing. The sequencing techniques combined with automation also advanced in the field of immunology and medicine. Forensics largely involves DNA sequencing technique. Sequencing technique helps in carrying out a large number of experiments in a very short period. Finished chromosome sequences also get detected accurately through high throughput sequencing and whole genome sequencing.

References:
[1] Human Molecular Genetics 3, Volume 3, T. Strachan, Andrew P. Read
[2] Medical Genetics, Lynn B. Jorde, John C. Carey, Michael J. Bamshad
[3] Essential Genetics, Daniel Hartl

© Copyright, 2018 All Rights Reserved.