Category: PCR Cloning

Applications of PCR Cloning Technology

PCR cloning technology can amplify trace amounts of DNA, generating millions of copies of a specific DNA sequence. PCR is highly sensitive, extremely specific, and high-yield, while also being easily reproducible, fast, and convenient, making it an amazingly powerful tool in molecular biology. With the rapid development of modern life science, PCR cloning technology has been more and more widely applied to various fields in biological research, medical research, virus detection, and the food industry.

PCR cloning Technology Applied to Gene Cloning

Gene cloning and Subcloning via PCR technology plays a significant role in cell biology research. PCR technology can generate millions of copies of a single-copy gene, amplifying a specific DNA fragment that might only be a few picograms. Compared to other gene cloning techniques, PCR omits several tedious processes for cloning of a particular gene fragment from genome DNA, such as enzyme digestion, connection, transformation, DNA library construction, gene screening, gene identification, and Subcloning.

PCR Cloning Technology Applied to DNA Recombination

In molecular biology, PCR cloning technology can be used to construct recombinant DNA molecules by inserting different sources of specific genes or DNA fragments into viruses, plasmids or other vectors in vitro. Recombinant DNA molecules are then imported into reporter cells to amplify and reproduce. After screening, the daughter cells that contain the target gene are further multiply to extract a large amount of DNA. Recombinant DNA technology can be applied to the Human Gene Project, valuable protein expression, gene diagnosis and therapy, genetic modification of animals and plants, and other research fields.

PCR Cloning Technology for Gene Quantification

PCR cloning technology can be applied to quantitatively determine the copy number of a target gene in a sample. The target gene and a single copy reference gene are placed in a tube for PCR cloning. The PCR product is then separated by electrophoretic separation technology and band intensity is observed. Alternatively, the 5’ end of the primer can be marked by a radionucleotide, after which the gene copy number can be determined by radioactivity quantification. PCR cloning technology can also be applied to quantitatively analyze mRNA and tRNA. It can even detect 1TIRNA, which is hard to detect even by Northern blot.

Alteration of endogenous genes and invasion of foreign genes can be threatening to human health. Regardless of whether or not pathogenesis is caused by genetic changes, as long as there is a pathogen, its corresponding existing nucleic acid can be found. With the development of PCR cloning technology and related technologies, PCR can be applied to infectious disease pathogenesis detection and diagnosis, tumor related gene detection, hereditary disease early diagnosis, bone marrow transplant HLA – D locus matches, and evolutionary theory analysis.

Synbio Technologies’s Syno® 2.0 platform can clone a target gene to any specific point on provided vectors without depending on restriction enzyme sites, and can quickly and cost-effectively fulfill a wide variety of client requests for myriad applications in synthetic biology.

PCR Techniques in Biology

Polymerase chain reaction, generally known as PCR and also referred to as in vitro DNA amplification, is one of the most widely used techniques in biology labs across the world.

Traditional DNA amplification method

Traditional DNA amplification involves the construction of a vector containing the target gene, followed by the transfer of the vectors into cells to amplify the target gene and then screening by probe. The process includes enzyme digestion, connection, transformation, culture, and probe hybridization. Although there are no technical difficulties for the traditional method, the complicated operation and long cycle length make it suboptimal as a way to amplify DNA. PCR is faster, more efficient, and cheaper, and has many advantages over traditional DNA amplification.

Advantage of PCR cloning

PCR was first theorized by Kary Mullis in 1983 and was later invented in 1985. PCR can amplify traces of DNA and generate millions of copies of a particular DNA sequence. Because of its high sensitivity, specificity, and yield, along with its reproducibility, speed, and convenience, PCR is now widely used in microbiology, medical science, agriculture, and many other fields. PCR has greatly simplified the process of molecular cloning, enabling researchers to much more easily analyze and identify genes of interest.

Basic principles of PCR cloning

At a temperature of 95℃, DNA denatures and yields single-stranded DNA molecules. The primer then binds to a complementary part of the DNA template. The temperature is then lowered to the optimum activity temperature, which is usually around 72℃. This activates DNA polymerase, which synthesizes a new DNA strand complementary to the DNA template in the 5’ to 3’ direction. The DNA template, primers, and polymerase are then thermocycled through denaturation, annealing, and extension steps, allowing DNA polymerase to replicate a target region of DNA by millions of times or more.

PCR has become an essential part of biology labs around the world, and is widely applied to gene cloning, genetic recombination, DNA sequence analysis, and gene quantification. PCR Cloning is also used in cancer gene detection and early diagnosis of hereditary diseases.

Our Syno® 2.0 platform can clone any target gene to any specific point on required vectors

without depending on restriction enzyme sites. We promise both accurate and speedy delivery of any client’s cloning requests.

The Methods of PCR Cloning

PCR Cloning is a technique used to amplify a specific region of DNA strand, and is used in almost every molecular biology lab in the world. PCR can be used on almost any DNA region, provided that suitable primers can be made. Synbio Tech provides one-stop PCR cloning services, including primer design.

PCR Primer Design Procedure

  • Acquisition of DNA sequence: For amplification of known DNA sequences, tried-and-tested primers can be found on the NCBI website. For unknown DNA sequences containing conserved sequence(s) from related species, primers should be designed according to the DNA or RNA of the conserved sequence(s).
  • PCR primer design: Many commercial software products and online tools are used to design primers. Primer Premier 5.0, the most popular primer design software, is both powerful and convenient to use. It can also contrast and comprehensively assess the best choice of primer according to their specificity.
  • Validation of PCR amplification: Once primer synthesis finishes, the accuracy of the primer can be predicted after gel electrophoresis of PCR product.

Notes About PCR Primer Design

  • The length of primer should be around 18-24bp. If the primer is too short, the primer specificity will be too low; if the primer is too long, it may cause base pair mismatches and may reduce the PCR amplification efficiency.
  • The GC% of the primer will affect the denaturation temperature (Tm). Tm should be around 55-80℃ and the annealing temperature difference between the upstream and downstream primers should be within 10℃. Usually, the GC% of primer should be between 40%-60% ,and the GC% difference between the upstream and downstream primers should be within 20% of each other in order to enhance primer specificity.
  • Repetitive structures or high similarity with the template sequence should be avoided as both may lead to possible base pair mismatches.
  • Secondary structure of primers may inhibit the PCR reaction, and should be avoided.

Synbio Technologies provide one-stop PCR cloning services, including primer design. Our Syno® 2.0 platform can clone the target gene to any specific point on a provided vector without relying on restriction enzyme sites, to best satisfy the cloning requirements of each client.

Synbio Technologies’ PCR cloning and Subcloning Technology

PCR cloning and subcloning technology, first developed in the 1970s, is now a staple in every molecular biology lab in the world. Cloning allows researchers to much more easily understand gene function at a deeper level, and greatly facilitates gene editing. PCR cloning and subcloning technology is not only revolutionary for the field of biology, but has profound implications on fields like agriculture, industry, and medicine as well.

PCR cloning technology

PCR cloning technology is similar to natural DNA replication, and contains three basic reaction steps: modification-annealing-extension. We can obtain a desired target gene sequence with appropriate primer design. PCR can then be used to amplify this gene sequence, preparing it for use in cloning.

Subcloning technology

In molecular cloning, target DNA is assembled into a vector plasmid through restriction enzymes and screening. In subcloning, a gene of interest is transferred from one vector to another. Both processes consist of several key steps, such as screening of the target fragment, cloning vector preparation, transformation/transduction of the product into cells, and screening for cells containing recombinant plasmids.

Both PCR cloning and subcloning technology can insert a target gene into a plasmid of choice in vitro through recombinant technology. The main forms of target gene transfer into a plasmid are transformation and transduction. This allows researchers to have an enormous amount of customization available to them when trying to study a gene of interest, making cloning and sub-cloning two extremely powerful tools in a molecular biologist’s arsenal.

With our proprietary Syno® 2.0 gene synthesis platform, Synbio Technologies can provide one-step services for gene synthesis, vector construction, PCR cloning, and subcloning. Customers only need to offer the sequence information, and we can help design amplification primers and clone the PCR products to the specific sites of the new plasmid. We also provide sequencing services in order to confirm that the correct product was accurately synthesized.

Gene Synthesis and Cloning

Gene Synthesis and Cloning is an integral part of many research pipelines. There are two popular methods of genetic cloning: DNA amplification by use of subcloning and polymerase chain reaction (PCR). The former needs more time and resource consuming, while the latter spends less time and cost effectively. Traditionally, subcloning was commonly used for amplification. But recently PCR has become much more effective and popular. The popularity of amplification by subcloning may have dwindled because it requires multiple steps. The process starts with the removal of the genetic sequence of interest using specific restriction enzymes. This same restriction enzyme is then used to open up the plasmid where the genetic sequence will be inserted into. This creates “sticky ends” which allow the genetic sequence to be more easily inserted. The genetic sequences is then added into a plasmid and sealed in place with DNA ligase. The plasmid is subcequently inserted into bacteria where the genetic sequence of interest is amplified and later extracted. This is a somewhat laborious process when compared to PCR. PCR has very simple steps: extracting the genetic sequence of interest and loading it into the machine. The sequence of interest is then rapidly amplified with a high quality output. The amplified sequence can then be used to in various research topics specific to the user. Amplification of these sequences were once restricted to only allow preexisting sequences to be extracted and amplified, but recent technology has allowed a small change in this.

The interesting aspect that Synbio Technologies offers is the ability to synthesize the genetic sequence of interest, as opposed to extracting the preexisting sequence from an organism. This technology is referred to as our Syno®2.0 Platform, which allows Synbio Technologies to successfully synthesize any desired genetic sequence, specified by the user. The lengths of these sequences can vary from a small as a few hundred base pairs to up to 150 Kb or even more in length. The Syno®2.0 Platform provides an extremely effective and revolutionary approach to genetic research. Traditionally, the sequence of interest must be present within a genome of an organism to then be extracted and later amplified. This platform allows us to synthetically create a sequence that does not need to be preexisting within any organism. The Syno®2.0 Platform allows us to move from a text file of the user specified sequence to the physical genetic constructs, bypassing the previous stipulation of extracting the preexisting sequence. The resulting synthetic products is then analyzed using Sanger Sequencing in order to verify and guarantee one hundred percent accuracy. Once successful, Synbio Technologies offers the option to clone this sequence as many times as necessary. This is done by use of either PCR or subcloning, both of which are extremely effective mechanisms to amplify the genetic sequence. The process of gene synthesis and later gene cloning allows Synbio Technologies to supply the researcher with a sufficient amount of genetic material needed within an efficient timeframe.

Synbio Technologies also offers PCR cloning if the requested sequence of interest is already present and does not need to be synthetically engineered. First the provided sequence will be verified using Sanger Sequencing in order to account for one hundred percent accuracy. The resulting verified sequence will then be put through our flexible and reliable pipeline of gene cloning. Using patent pending, Syno®2.0 Platform and Clone®3.1 system, Synbio Technologies is able to achieve inserting any sequence of interest into any site of a vector specified by the user. This allows the user to specify which restriction enzyme to use, both for the extraction of genetic sequence, as well as the restriction enzyme used on the plasmid. These wide range of possibilities allows the researcher to design a location within a specific vector in order to amplify the sequence of interest. This specificity is something that Synbio Technologies is very proud of, allowing us to adhere to any requests that the user might have while also providing a high quality output in the process.

At Synbio Technologies we pride ourselves as being one of the leading companies in the biotechnology industry, especially when referring to gene synthesis. We offer both accurate and time effective approaches to gene synthesis and gene cloning. The genetic sequence of interest will be Sanger Sequenced and verified for both genetically synthesized sequences as well as sequences provided by the user. This allows us to verify that the sequence, as well as the resulting amplified sequences, are extremely accurate before shipping the resulting product to the user. If the DNA sequence of interest has not yet been sequenced with high quality, we will conduct sequence validation in order to verify the accuracy before amplification. In addition to the accuracy and time efficiency, Synbio Technologies offers competitive prices for both gene synthesis and cloning of your sequence of interest. With competitive prices, along with the verified accuracy and time efficiency, Synbio Technologies is ready to offer customers a unique pipeline to utilize and rely on when conducting various types of genetic research that including gene synthesis and gene cloning.