Gene cloning and vector construction are some of the most frequently used techniques in molecular biology. Short DNA fragments or whole genes are inserted into plasmids or virus chromosomes, replicated in a bacterial or eukaryotic host and subsequently isolated allowing for downstream sequencing/characterization of the gene of interest, or study of gene expression/protein production. Whatever your requirements, Meridian has an extensive range of cloning reagents to expertly prepare your targets plus a selection of competent cells to suit your specific application, all offering efficient transformation with rigorous quality control for reproducible results.
Basic Steps of Cloning
There are many approaches available for cloning DNA, most of which follow this basic workflow:
- Isolation of target DNA - typically referred to as an ‘insert’
- Ligation of insert into chosen vector, creating a recombinant molecule - vectors are typically bacterial plasmids, or viral chromosomes
- Transformation of recombinant molecule into a suitable host for propagation - typically referred to as a ‘competent cell’
- Screening and selection of propagated hosts that contain the recombinant molecule for subsequent isolation of the insert
Preparation of DNA inserts can utilise genomic DNA that is fragmented with restriction enzymes, cDNA and/or other PCR products.
Ligation of the fragments or inserts into the chosen vector requires the action of an enzyme and T4 DNA ligase is the most common choice. Meridian has formulated a quick-stick T4 DNA ligase with specially developed buffer to improve enzyme activity and increase efficiency of the ligation step.
Successful vectors must be able to incorporate the inserted DNA fragment and maintain replicative functionality. Suitable hosts must be able to take in the vector, maintain viability and continue to replicate, resulting in the propagation of clones containing the insert of interest. Transformed cells are typically at a growth disadvantage and as such, vectors will contain a selective characteristic such as antibiotic resistance, so that only clones containing the vector will grow on selective media. An additional characteristic of competent cell hosts is their mechanism of indicating a positive insert clone. All competent cells supplied by Meridian contain genes for X-gal blue/white colony selection.
Cloning Strategies and Choosing Vectors
Choosing an appropriate cloning strategy and vector/host will depend on a number of different factors:
- Insert size
- Desired copy number after transformation/ intended downstream application (purification/sequencing or protein expression/production)
- Number of cloning sites in vector/compatibility with restriction sites in insert
- Compatibility of resulting protein in host (if insert is whole gene fragment)
- Selective mechanisms of competent cells
DNA Source and Insert Preparation
If using genomic DNA, restriction endonucleases (REs) are used to create fragments of a size that can be cloned – typically REs recognize 6-8 consecutive bases.
Most REs leave base overhang or “sticky-ends” which allow for more efficient ligation with T4 DNA ligase. Meridian vectors contain multiple cloning sites for compatibility with majority of REs. If using genomic DNA from eukaryotic cells, it will likely be heavily methylated, thus the chosen vector/host must be mcr mutant in order to not degrade the insert – Meridian offer a range of such cells for these situations.
Many cloning applications utilize PCR products of specific genes or areas of interest and these can either be blunt-end (if using a DNA polymerase with proof-reading capability), or have an A-overhang if using non-proofreading Taq or Tht polymerases. The addition of the nucleoside adenosine to the 3’-end of amplicons during PCR is a characteristic that is often utilized in cloning workflows to avoid additional use of REs and many vectors including a compatible T-overhang for streamlined and efficient ligation.
Plasmid, Phage or F’- episome Vectors
Bacterial plasmids are a common choice for cloning vectors, as they contain all genetic elements for replication and are easily isolated from transformed cells for downstream applications.
Typically plasmids can effectively carry an insert between 5-10 kb and some are specially formulated to carry larger inserts up to about 15 kb. For applications that require even larger inserts, phage vectors are typically employed. Lambda bacteriophage vectors can typically carry up to 24 kb and have 10-100x greater transformation efficiency in E.coli competent cells, however downstream isolation is more complicated compared with plasmid isolation.
Some plasmids can also be incorporated into the bacterial host chromosome and these are referred to as episomes. Filamentous phage can package single-stranded DNA inserts and F’ episome hosts can incorporate these, typically with high transformation efficiency (>50%). Single-stranded DNA clones are useful for downstream applications such as site-directed mutagenesis or sequencing with dideoxynucleotides.
Competent Cell Characteristics
The majority of competent cells on the market are known as chemically competent, treated with salt wash that disrupts the membrane for easy uptake of the vector.
The advantage of using chemically competent cells is that no specialized equipment is required, however cells that are treated with an electroporation device – electrocompetent cells - have higher transformation efficiency.
All of the competent cells from Meridian are recombination and endonuclease deficient, improving vector stability and transformation efficiency, plus they are all compatible with X-gal mediated blue/white colony selection. In blue/white screening, vectors containing an insert disrupt β-galactosidase activity and have no ability to convert X-gal medium to blue, thus positive clones will appear as white colonies, while transformed cells without an insert grow blue.
DNA cloning forms the precursor to many molecular biology workflows, providing a mechanism to produce large quantities of particular segments of DNA which can be used for a variety of research and applied science purposes.
The human genome sequencing project utilized large-scale DNA clone libraries produced with random fragmented genomic DNA that was subject to several rounds of dideoxy sequencing. This approach, known as ‘shot-gun’ sequencing, allows multiple, overlapping reads to be assembled into continuous long-reads that ultimately enabled the full genome sequence to be determined. Gene clone and DNA clone libraries continue to be an effective an efficient method to characterize populations, genomes, or specific genes of interest with downstream sequencing techniques.
Cloning workflows can also be employed to support the study of the structure and biological activity of genes, such as using site-directed mutagenesis in an effort to alter gene property or function. Synthetic gene copies containing the desired mutation are used as inserts and cloned to produce multiple copies of the mutated gene for downstream applications. DNA cloning can also be used for protein production, although in practice providing the appropriate conditions for active protein production is complex due to the specific requirements for folding, stability and transportation.