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.

Molecular testing helps to detect gene mutations

The basic unit of the heredity known as the gene plays a crucial role in disease and inheritance. If it gets mutated at wrong sites, it passes on the undesired traits to the population. Genetic diseases arise due to an underlying mutation in the gene. The effect of all mutations may not necessarily be a disease. However, the mutations occurring at wrong sites, lead to altered products such as proteins and enzymes having altered functions leading to genetic disorders. There are many examples of genetic disorders in humans. They follow different patterns of inheritance. For example, the BRCA gene mutation leads to breast cancer. Similarly, mutations in the mitochondrial DNA follow the maternal pattern of inheritance.

Image 1: Molecular testing

The genetic disorders are either present by birth or may develop during later stages of life. Most of them are congenital, meaning, their occurrence is by birth. With the help of genetic testing, it is possible to detect them before birth. Importance of genetic testing lies in the prevention of genetic disorders. It helps in preventing the birth of babies who may suffer later on. It helps in decision making and genetic counseling. Genetic testing also reveals types of mutations. We can understand the chances of getting a disease in the future. Thus, the field of genetic testing is advancing further. With the help of genetic testing, it is possible to detect disease genes in a family.

The field of diagnostics involves detecting a disease, the stages of the disease, and the causative agent. Genetic testing helps to detect the gene involved in mutation, the type of mutation, chances of inheriting a disease, and whether the disease will occur in the future. It mainly targets a population with a family history of diseases. The purpose of genetic testing is to detect any mutation before the birth of the baby. There are three important applications of genetic testing such as prenatal diagnosis, newborn screening, and carrier detection. Prenatal diagnosis checks whether the fetus is at risk. Two important procedures involved in prenatal diagnosis are amniocentesis and chorionic villus sampling. These two procedures being very painful and risky led to the development of non-invasive prenatal testing (NIPT). It involves testing cell-free DNA that floats in the maternal plasma. The prenatal tests can detect gene mutations and chromosomal abnormalities. The embryos containing mutated genes lead to serious genetic diseases. Genetic testing helps to detect mutations in the embryonic genes. Newborn screening detects mutations in the newborn babies.

For example, the PKU test detects mutations for phenylalanine that causes phenylketonuria. Carrier detection helps to find out whether the individuals are carriers for a gene mutation. The example involves sickle cell anemia. The carriers are known as heterozygotes. DNA molecular testing determines the molecular nature of mutations associated with the disease.

Molecular testing methods include RFLP and PCR Analysis:

1.     RFLP Analysis:

It is a commonly used genetic test. It involves analyzing restriction fragment length polymorphisms. It arises due to a different pattern of restriction sites. The RFLP analysis involves three main steps such as restriction digestion, agarose gel electrophoresis, and hybridization. The DNA is fragmented using restriction enzymes. The fragments obtained from restriction digestion are separated based on the fragment size using AGE. The gel containing separated fragments is placed in a buffered solution to transfer the DNA to a membrane filter. The DNA on the membrane filter hybridizes with the probe having a complementary sequence.

The product of restriction endonuclease action on a DNA is known as a restriction digest. The restriction fragment length polymorphism is nothing but a variant in DNA banding pattern of an electrophoresed restriction digest. It occurs when the length of the fragment varies between the individuals. Each length of a fragment is an allele. The DNA shows restriction enzyme cleavage site at a place in the genome of an individual which is missing is another individual.

Image 2: RFLP analysis

An example of RFLP analysis involves detection of sickle cell gene by Dde I RFLP. Abnormal hemoglobin results into sickling of red blood cells in sickle cell anemia. It arises due to a beta-globin gene mutation. It involves a single base-pair change of AT to TA leading to single nucleotide polymorphisms. The sixth codon of the beta-globin thereby changes from GAG to GUG. Instead of glutamic acid insertion in a polypeptide, valine gets added. This mutation results in RFLP for restriction enzyme Dde I. This enzyme has a restriction site where the fourth base pair changes.

In normal individuals, there are three Dde I sites in the beta-globin genes. One of the Dde I sites is situated upstream while the other two sites are present in the coding regions. In the individuals having sickle cell anemia, the beta-globin genes have only two Dde I sites instead of three. The mutation removes the Dde I site present in the middle. RFLP analysis helps to study individuals affected with sickle cell disease and compare the results with the normal individuals.

In a normal individual, the fragments hybridized with the probes reveal two fragments after visualization. One fragment shows 175 base pair size. The other fragment shows 201 base pair size. In persons with sickle cell disease, the hybridized DNA, when visualized, reveals only one fragment of 376 base pair.  The DNA obtained from heterozygotes reveals three bands such as bands with 376, 201 and 175 base pairs respectively. Let us consider another example involving mutations in the DNA flanking the gene. The DNA flanking the gene may be very far away from such as PKU genes. In such cases, the detection of mutation relies on flanking RFLP.

2.     PCR Analysis:
     

Image 3: Polymerase chain reaction

    For PCR analysis, the sequence information helps in designing the primers. Polymerase chain reaction or PCR is nothing but amplifying the desired DNA under controlled conditions. A common test involved in the PCR analysis is Allele-specific oligonucleotide hybridization (ASO). A short single-stranded DNA capable of being synthesized in a test tube is known as an oligonucleotide. It is less than 50 nucleotides in length. These small DNA molecules are capable of hybridizing. They form a complementary base pair with other DNA molecules. A single mismatch may not allow an oligonucleotide to hybridize, thereby discriminating between the two alleles of an SNP.

Consider an example of ASO hybridization. The GLC1A gene mutations lead to ophthalmic disorders. A common example includes open-angle glaucoma. It is a condition in which there is an increase in eye pressure. It may lead to total blindness due to neglection. One of the GLC1A gene mutations involves CG to TA change. Thus the codon changes from CCG (encoding proline) to CUG (encoding leucine). The DNA sequencing of the heterozygotes revealed both wild-type and mutant alleles. Agarose gel electrophoresis helps to separate the fragments. The DNA denatures into single strands for ASO hybridization. The detection becomes easy through radioactive signaling. There is one more concept associated with ASO. It is known as reverse ASO hybridization. The method uses radioactively labeled PCR products as probes for hybridization with many different ASOs. It can detect many mutations.

Sometimes it might be difficult to find some genes in the genome. The gene may be cloned but not sequenced. Molecular testing tools may not be available for such genes. Sometimes a single molecular test may be futile if the gene has many mutations. RFLP and PCR can better detect single base pair mutations or genes with only a few mutations. In case of multiple mutations, designing of a subset for known mutations may help.

References:
[1] RFLP analysis, Thermofisher Scientific
[2] Molecular Biology Techniques: An Intensive Laboratory Course, Walt Ream, Katharine G. Field
[3] Ana Techniques in Biotechnology, Goutam Bhowmik

© Copyright, 2018 All Rights Reserved.

SNP analysis

Single base pair changes in the DNA may or may not be responsible for a disease. Not all mutations are harmful. It is true since some changes in the DNA also prove beneficial. Adaptation, evolution, variation, and many other phenomena occur due to single base pair changes in the DNA. Hence, it is important to study both harmful and harmless mutations. A better way to describe single base pair changes involves the following way. The single base pair positions in the DNA are also known as snips or the single nucleotide polymorphisms or SNPs. The single nucleotide polymorphisms involve those types of single base pair changes which show different sequence alternatives. The least frequent allele in a normal individual shows an abundance of 1% or greater. SNPs also encompass low penetrance, quantitative trait loci, and the alleles associated with the risk. These properties also occur in a normal individual to some extent.

Sometimes, the presence of an SNP in a gene disrupts a gene function. Hence, SNPs lead to one base pair change in the DNA. The SNPs effectively serve as markers. Some references point out the comparison of the SNPs with the point mutations. They arise due to the alteration in the sequence of the DNA. It involves a single nucleotide base change, insertion or a deletion. However, the SNPs and point mutations differ only in their frequencies. Point mutations arise as a result of the occurrence of 1% or less than 1% of the variation in a population. Hence, the low-frequency mutations do not serve as good markers. The SNPs occur in the various regions in a genome. Different SNPs exist in the coding as well as the non-coding regions in a genome. The coding regions, also known as exons, code for the specific amino acids. The non-coding regions, known as the introns, do not get involved in coding and hence get spliced out before the translation occurs.

The concept of haplotyping involves grouping the subjects by haplotypes or particular patterns of the sequential SNPs found on a single chromosome. The classification of a single-base position involves three main types. If a chromosome consists of a normal allele, the given single-base position is homozygous for a wild-type base. If there is an altered allele in each chromosome under review, the given single-base position is homozygous for the SNP-base. If one chromosome has a normal allele and the other has an abnormal allele, the condition exhibits heterozygosity. Hence, SNPs serve as the disease gene markers. Every gene may alternatively show the presence of the multiple SNPs. If a coding region involves a smaller percentage of the SNPs, less sequence variation prevails there. The coding sequence codes for an amino acid conferring to a protein structure or a function. The SNPs within this region may alter the protein function, its structure or both. SNP analysis is highly advantageous. SNPs serve as efficient markers for gene mapping. They allow a systematic identification of alleles in normal and diseased individuals.

Image: SNP analysis

Detection of SNPs:

SNPs constitute the most common types of DNA polymorphisms. Most of the SNPs occur in the non-coding regions. Hence, they are known as non-coding SNPs. The coding regions also have SNPs and are also known as cSNPs or coding SNPs. SNPs occurring in the regulatory sequences are known as rSNPs. They also refer to anonymous SNPs occurring in the junk DNA. Most of the cSNPs cause missense mutations. The SNPs also affect the restriction sites. The Southern blotting or PCR techniques help in detecting the SNPs at the restriction sites. The DNA sample first gets a treatment with the restriction enzyme. The resultant fragments get separated using gel electrophoresis. After obtaining the bands of different sizes on the gel, the DNA present on the gel gets transferred to the membrane filter using a Southern blot apparatus. After hybridization with a probe, the bands get visualized using autoradiography technique. Hence, homozygotes and heterozygotes for SNP alleles get detected.

Another way to detect SNPs involves PCR amplification. The PCR technique or Polymerase Chain Reaction technique involves isolation of DNA, amplification, digestion with the restriction enzymes, and gel electrophoresis. Another technique involves allele-specific oligonucleotide hybridization (ASO). Small oligonucleotides help in detecting the single nucleotide polymorphisms. Other techniques for typing thousands of SNPs include DNA microarrays or DNA chips or Gene Chips.

DNA Microarray:

It is an ordered grid of DNA molecules with known sequences. They get fixed at a particular position on a silicon chip or a glass or membrane filter. The automated machines help in depositing the microspots in a known location. The oligonucleotide arrays have a small size similar to a postage stamp. The examples of DNA microarray include GeneChips^®. There is no labeling to the DNA molecules fixed on the slide or a glass or a silicon chip. However, the target DNA involves labeling. The target DNA labeling uses fluorescent tags such as a Cy3 dye. After hybridization, laser scanning helps to detect fluorescent molecules and study the pattern across the array. Cancer genomics experiments frequently use DNA microarrays for detecting mutations. Hence, the DNA microarray helps in detecting SNP alleles in an individual. There are two types of spots observed across the array. A green spot indicates a green-labeled target DNA hybridization. A red spot indicates a red-labeled DNA hybridization. The shades of yellow spots indicate hybridization depends on the spot color.

GeneChip technology helps in assessing a large number of SNP alleles. It is easy to map the genes associated with the complex traits using DNA microarray.           

References:
[1]Forensic DNA Typing: Biology, Technology, and Genetics of STR Markers, John M. Butler
[2] Human Genetics and Genomics, Includes Wiley E-Text, Bruce R. Korf, Mira B. Irons
[3] SNPs, Genetics Home Reference
[4] DNA Microarray, Wikipedia             
     

© Copyright, 2018 All Rights Reserved.