PCR and recombinant DNA technology are essential for different branches of biology including biotechnology and genetic engineering. They are used to replicate and manipulate genes and alleles for various purposes. Despite their utilization in DNA multiplication, they are distinct from each other. Keep reading to learn all the differences between PCR and recombinant DNA technology.
Comparison Table
| Characteristics | PCR | Recombinant DNA |
| Definition | Gene amplification | DNA isolation and manipulation |
| Mechanism | Amplification of gene in PCR tubes | DNA fragments into a suitable vector |
| Enzymes | DNA polymerase | Restrictive enzymes |
| Stages | Denaturation, annealing, extension | genetic isolation, gene removal, inc. gene copies |
| Applications | Detecting microbes | Pharmaceutical preps |
What is PCR?
Polymerase chain reaction (PCR) is a technique to generate bulk copies of target DNA and genes from a small strand. This technology enables a scientist to produce millions or billions of DNA copies per requirement for studying and analyzing. In simple words, you can get bulk information from small data for genetic studies. It is widely used in molecular biology and biochemistry.
Steps of PCR
To carry out a successful PCR procedure, you need the following components:
- DNA or RNA specimen of interest that needs to amplify
- A DNA primer which is a brief piece of single-stranded DNA that encourages the production of a complementary strand of nucleotides
- Taq DNA polymerase to aid in creating complementary strands
- A mixture of the nucleotides adenine (A), thymidine (T), cytosine (C), and guanine (G)
No matter what type of DNA sample you use for any kind of PCR, the underlying principles of PCR remain the same. Denaturation, annealing, and extension are the steps the thermal cycler uses to process the solution. Thermal cycling, a machine-assisted heating and cooling process, is a PCR component. The steps of PCR include:
Denaturation: The first step involves heating sample material from 95 to 98 degrees Celsius. It denatures the double-stranded DNA and divides it into two single strands.
Annealing: The second step involves lowering the temperature to between 55 and 65 °C so the primers can anneal or attach to specific DNA sequences at each end of the target sequence, commonly known as the template.
Extension: The temperature is often raised to 72°C in the third step, enabling the DNA polymerase to extend the primers by adding dNTPs to generate a new strand of DNA, doubling the amount of DNA in the reaction.
The process is then automatically repeated 35 to 40 times using a thermal cycler, which recounts the heating and cooling cycles. The resulting DNA sequence is doubled with each heating/cooling cycle the cycler does. Therefore, you can amplify a tiny piece of DNA from one sample to create millions of copies after 35 doubling cycles.
Applications of PCR
This revolutionary technology has a wide range of scientific applications in almost all fields of biology. Some of the applications that are making revolutions for humanity are below.
- To examine DNA samples from crime scenes, forensic labs employ it. Clinical healthcare laboratories utilize PCR to identify virus-infected people.
- For usage in the production of medications like antibiotics and vaccines, pharmaceutical research labs utilize it to replicate and analyze DNA and RNA samples.
- You can use PCR in microbial ecology, environmental sciences, microbiology, especially when finding microorganisms in the environment is necessary.
- Based on each person’s DNA data, a market has emerged (using PCR) that provides customers with specialized goods and services.
- Many Food and Agriculture Biotechnology researches are using PCR to meet the demands of a growing population.
What is Recombinant DNA Technology?
The fusion of DNA molecules from two different species is known as recombinant DNA technology. DNA molecules are recombined in host organisms to create new genetic combinations that can be used in science, medicine, agriculture, and industry.
With a number of enzymes and numerous laboratory procedures, it is possible to manipulate and isolate desired DNA segments through recombinant DNA technology. Using this technique, one can join (or splice) DNA from several species or produce genes with novel functions.
Recombinant DNA is the term used to describe the resultant copies. Recombinant DNA is commonly propagated in bacterial or yeast cells, whose biological machinery copies the modified DNA alongside its own.
Steps of Recombinant DNA Technology
DNA sequencing originating from several sources is known as recombinant DNA. The steps in recombinant DNA technology are
Genetic Material Isolation (DNA): The cell must split open to release DNA and other macromolecules, including types of RNA, proteins, carbs, and lipids, because the membrane shields the DNA.
Therefore, the cell wall is disintegrated to liberate the genetic material to remove it. Cell wall digesting enzymes, such as lysosomes for bacterial cells, cellulose for plant cells, and chitinase for fungal cells, can be used to achieve this. Since genes are known to be held on lengthy DNA molecules entangled with proteins like histones, ribonuclease can remove RNA, while protease can be employed to remove proteins.
DNA Cutting: Restriction enzymes are used for this purpose, which allows the necessary DNA fragments to be produced by cutting DNA at specific recognition sequences.
The restriction enzyme cuts the DNA after purifying it under controlled and ideal circumstances. A restriction enzyme digestion is monitored using agarose gel electrophoresis. DNA, as it is a negatively charged molecule, flows toward the cathode from the anode. The vector is also used in this process again.
Combining DNA Fragments: The same restriction endonuclease is used to cut the vector DNA (plasmid DNA, for example) and alien (foreign) DNA carrying the desired gene, resulting in complementary sticky ends. Restriction enzymes cleave DNA during the restrictive digestion process.
A recombinant (chimera) DNA is created by annealing the complementary sticky ends of two DNAs with the aid of the DNA ligase enzyme (rDNA). The ligase joins two DNAs by forming new sugar-phosphate linkages. Sticky ends ligation, blunt-end ligation, and homopolymer tailing are all techniques for assembling the DNA fragments produced by a cloning vector.
DNA Insertion into Host Cell: At this point, the recombinant DNA is inserted into the host, and the procedure is called transformation. In this transfer procedure, various vector-based and vectorless techniques are utilized.
Transformation, transduction, and vectorless gene transfer are three methods for introducing recombinant DNA into the host cell. Selecting and separating the transformants from the non-transformants is facilitated by culturing the cell mixture in an antibiotic-containing medium. The different selectable markers are crucial for distinguishing recombinants from non-recombinants.
Collection and Testing of Altered Cells: The desired protein is the primary goal of most recombinant technologies. Recombinant DNA must therefore be expressed.
The expression of the introduced gene is the process by which the recombinant DNA containing the desired gene manifests itself by creating a protein.
One must keep the ideal conditions for inducing the expression of the target protein after the gene of interest has been cloned, and one should think about mass manufacturing it. A “recombinant protein” has a gene for a protein produced in a heterologous host. Any techniques, including insertional inactivation, blue-white screening, nucleic acid hybridization, and immunological approach, can be used to select and screen altered cells.
Applications of Recombinant DNA Technology
The most widely adapted applications of DNA recombinant technology include:
- DNA fingerprinting in forensics
- Diagnosis of genetic diseases and disorder
- Gene therapy to improve genetic diseases
- Production of hepatitis B vaccine
- Synthesis of human insulin using Recombinant DNA technology
Difference between PCR and Recombinant DNA Technology
Definition
PCR
PCR synthesizes multiple copies of specific DNA of interest from a small fragment through amplification.
Recombinant DNA Technology
rDNA refers to fusing DNA molecules from other organisms and inserting those DNA molecules into a host organism to create novel genetic combinations.
Mechanism
PCR
In PCR, the amplification of small DNA fragments is carried out under in vitro settings in PCR equipment inside PCR tubes.
Recombinant DNA Technology
On the other hand, recombinant DNA technology entails inserting DNA fragments with acceptable gene sequences from several sources using the suitable vector.
Enzymes
PCR
PCR uses DNA polymerase as a crucial enzyme for creating complementary strands.
Recombinant DNA Technology
Whereas, restrictive enzymes play a vital role in recombinant DNA technology.
Steps
PCR
The three main stages of PCR take place at three different temperatures: denaturing of double-stranded DNA at 94 °C, annealing primers at 68 °C, and strand elongation at 72 °C.
Recombinant DNA Technology
Conversely, in recombinant DNA technology, the significant steps are genetic material isolation, removing the gene at the points of recognition, using the polymerase chain reaction to increase the gene copies (PCR), DNA molecules joining together, recombinant DNA insertion into the host.
Applications
PCR
PCR is used in detecting microorganisms and parasites like bacteria, fungi, and molds in microbiology due to its fast and precise technology.
Recombinant DNA Technology
At the same time, recombinant DNA technology is employed in the pharmaceutical industry to produce insulin.
The Bottom Line
PCR and recombinant DNA technology play a major role in genetic engineering, microbiology, biochemistry and molecular biology. They help researchers study, isolate, and modify DNA to alter an organism’s genotype and phenotype. While PCR has revolutionized rDNA, there are many differences between PCR and recombinant DNA technology. They differ in their mechanism, steps, and the enzymes involved. Scientists are working to find a cure to genetic diseases through genetic engineering.
Jeannie has achieved her Master’s degree in science and technology and is further pursuing a Ph.D. She desires to provide you the validated knowledge about science, technology, and the environment through writing articles.
