What are the basic features of a plasmid?
Essentially, plasmids are small, circular molecules of DNA that are capable of replicating independently. As such, they do not rely on chromosomal DNA of the organism for replication. Because of this characteristic, they are also referred to as extra-chromosomal DNA.
What are the features of plasmid being used as a vector?
Origin of replication (on), a selectable marker, sites for restriction endonuclease and its size, all are important features required to facilitate cloning into a vector. A good DNA vector should be able to replicate autonomously in the host cell, for which it needs to have an origin of replication site (ori).
What are the necessary components of a plasmid used in cloning?
components of plasmid cloning vectors:
- origin of replication (ori) site where DNA replication is initiated.
- marker genes for selection and/or screening.
- Unique restriction endonuclease (RE) sites. – allow inserts to be cloned in specific sites on plasmid.
- transmissability.
- Promoters for gene expression.
How are plasmids used in gene cloning?
Plasmids are often used in gene cloning as vectors to carry genes. The plasmid is isolated and treated with the same restriction enzyme as the target gene. The plasmid will mix with the target gene and recombinant DNA molecules are produced. The recombinant plasmid is taken up by the bacterial cell.
What are the basics of cloning?
The basic cloning workflow includes four steps:
- Isolation of target DNA fragments (often referred to as inserts)
- Ligation of inserts into an appropriate cloning vector, creating recombinant molecules (e.g., plasmids)
- Transformation of recombinant plasmids into bacteria or other suitable host for propagation.
What are the steps to cloning DNA?
Steps of DNA cloning
- Cut open the plasmid and “paste” in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA).
- Insert the plasmid into bacteria.
- Grow up lots of plasmid-carrying bacteria and use them as “factories” to make the protein.
What is PCR cloning?
PCR cloning is a method in which double-stranded DNA fragments amplified by PCR are ligated directly into a vector. TA cloning and blunt-end cloning represent two of the simplest PCR cloning methods. Their choice depends upon the nature of the vector and the type of PCR enzymes used in cloning.
Is PCR needed for cloning?
PCR cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than traditional cloning methods can accommodate. It allows for the cloning of DNA fragments that are not available in large amounts. Early PCR cloning often used Taq DNA Polymerase to amplify the gene.
What is the difference between DNA replication and cloning?
The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.
Does PCR have any limitations?
Although PCR is a valuable technique, it does have limitations. Because PCR is a highly sensitive technique, any form of contamination of the sample by even trace amounts of DNA can produce misleading results (Bolognia et al, 2008; Smith & Osborn, 2009).
Why did PCR fail?
Usually the first thing researchers do is blame a faulty enzyme or reagent when an experiment fails but with PCR this is actually less likely to be the cause for failure. More often deeper internal problems such as primer design, thermocycler parameters, or nonspecific binding to other template sequences are the cause.
What are the advantages and disadvantages of using PCR?
Table 1
| Advantages of PCR | Disadvantages of PCR |
|---|---|
| Shown to be more cost-effective with selective use than culture and staining | Becomes less cost-effective when performed with a multi-organism PCR approach |
| Increased ability to detect less common organisms such as viruses | Supply costs, machinery fees, training expenses |