Tag: subcloning

Molecular Biology Evolution

Since molecular biology was first established, in the 1930s, the complexity of various biological systems have been explored in order to better understand these systems. As years went by, and our understanding of these systems increased, the information that fell under the, large and ever-growing, umbrella of “molecular biology” became more specific and eventually subdivided by the particular sub-field being studied. Some of these sub-fields include: genetics, proteomics, and cytology. Many of these sub-fields have become extremely popular and very well studied since molecular biology was first established.

Genome Project and Molecular Biology

In the more recent past, there has been an influx of quantitative research within molecular biology. This groundbreaking research has completed the connection between two popular sub-fields of molecular biology: computer science through use of bioinformatics and computational biology. This was most notably seen with the success of the Human Genome Project (HGP). The Human Genome Project allowed for over three billion nucleotide base pairs of euchromatic genome to be sequenced and presented as a reference. This was an important milestone of molecular biology which allowed the human genome to go from the analog world of biology into the digital world. This was a giant step, within the field of molecular biology, which caused a domino effect that resulted in thousands of human genomes to be sequenced with increasingly lower cost.

On 2rd June 2016, the Genome Project-Write (GP-write) announced a continuation of genomics research through multiple molecular biology tools. These multiple molecular biology tools included gene synthesis and genome editing technologies. These technologies are utilized to synthesize and test large portions of many genomes stemming from microbes, plants and even animals. GP-write’s commitment to furthering our understanding and effectiveness of these technologies has the possibility to improve research and development of the topics of life sciences, new bio-based therapies, and nutrition.

New era of molecular biology

Even as these recent accomplishments in molecular biology unfold, a new problem seems to be stemming from them. The problem that has come about comes in the form of designing and even artificially synthesizing new life.

A recent study has proven the ability to accomplish a complete chromosomal transplantation from one cell to another. After the transplant has been successfully conducted, the chromosome can then be activated to conduct various genetic activity. We can then utilize specific enzymes, digestive proteins and other substances within these cells. This combination will result in the cell’s loss of original features and a totally new species.

Due to the crisis of resource shortage critical to human sustainability partnered with the ever-increasing human population, there is a need for us to seek effective approaches for sustainable living. The furthering of our knowledge on biological systems through these technologies would have many positive effects on successfully creating a sustainable habitat. These positive effects would come as a result of better understanding of the physiology of cells, developing new molecular medicines, as well as generating sustainable energy sources, such as biofuels. All of these will contribute to a more successful living environment through the use of molecular biology.

One molecular biology technology, gene synthesis, has slowly become better understood over the past ten years and has drastically increased our capability of editing and synthesizing genes of interest. Synthesizing DNA artificially is very difficult and increases in difficulty when attempting to synthesize long genetic sequences. This is because the longer the sequence being generated is, the higher the possibility of generating errors. Therefore, a new method is required to successfully conduct gene synthesis and correct for all mistakes generated when the sequence was being synthesized.

As one of the leading companies in the field of synthetic biology, Synbio Technologies has unique proprietary GPS platform on the basis of genotype, phenotype and synotype. We can provide excellent molecular biology services including: plasmid DNA preparation, PCR cloning and subcloning, site-directed mutagenesis and vector construction. We have the ability to generate sequences that are de novo, meaning that the genetic sequence is not preexisting within any organism in biology. We also have the ability to generate sequences up to and including 200kb in length in addition to complex gene products and structures. Synbio Technologies prides itself in our ability to use these molecular biology technologies to better suite our customer’s needs when conducting various types of research.

Molecular Biology Related Services

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.

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.

Four Steps of Subcloning Technology Protocol

Subcloning refers to the technique of re-cloning a DNA fragment from one vector to another, so that we can more easily perform analysis, transformation, and recombination of the target gene(s). Subcloning is an important tool in any molecular biologist’s toolkit, helping to elucidate the function of a target gene and to easily analyze its phenotype. There are four steps in the subcloning process: obtain the target fragment, connect enzyme vector and target fragment, transform in host cell, identify and screen.

  1. Subcloning Technology Protocol- obtain the target fragment
    • Find the target fragment in a gene library
    • The cDNA sequence was reverse transcribed with mRNA as a template through PCR, so we can obtain the target fragment from the cDNA library
    • Cut the DNA into many fragments with restriction enzymes and introduce into cells; we can screen for cells containing the target gene(s)
    • Synthesize the gene sequences in vitro
  2. Subcloning Technology Protocol- connect enzyme vector and target fragment

    3. Choose an appropriate restriction enzyme to cleave the target fragment from the vector. Cleavage in this way usually generates symmetrical cohesive ends, non-symmetric cohesive ends, or blunt ends. For subcloning, non-symmetric cohesive ends are preferred.

  3. Subcloning Technology Protocol- transform into host cell

    4. The two most common methods of transforming the target fragment into cells are transformation / transfection and transduction. In the first method, a recombinant plasmid or phage is simply transformed into a treated host cell; in the second, a host cell is transducted with a virus harboring exogenous DNA. In general, transduction is more efficient than transformation.

  4. Subcloning Technology Protocol-identify & screen

    5. Vectors with recognizable genetic markers can be used to help distinguish and separate cells transformed with recombinant DNA. For example, color can be used as such a marker, as the color of a colony may change in some vectors with exogenous genes. Other effective and widely used screening methods also exist, such as screening with drugs or with selective media.

With our Syno®2.0 gene synthesis platform and experienced technical team, Synbio Technologies provides one-stop subcloning services including target gene synthesis, vector construction and transformation, identification, and screening.

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.