12 Questions
What is the main purpose of molecular cloning?
To construct recombinant DNA molecules
Which type of cloning methods are now typically used for larger scale projects?
Ligation-free and recombination-based methods
What is a common application of molecular cloning?
Generation of genetically modified organisms
What is the first step in molecular cloning experiments?
Integrating foreign DNA into a suitable vector
Which factor determines the choice of a cloning vector in molecular cloning experiments?
The size of the foreign DNA to be cloned
What host organism is typically used in molecular cloning experiments?
E.coli
What is the purpose of a selectable marker in a plasmid-based cloning vector?
To allow for positive selection of transformants
Why can PCR be directly performed on prokaryotic genomic DNA (gDNA) but not on eukaryotic gDNA?
Eukaryotic gDNA contains introns that interfere with PCR amplification
What is the function of a cloning site (MCS) in a plasmid-based cloning vector?
To allow insertion of foreign DNA
What are the components typically found in a PCR Master Mix?
DNA polymerase, dNTPs, MgCl2, and reaction buffer
Why is reverse transcriptase used when working with eukaryotic DNA targets?
To convert mRNA to cDNA and remove introns
What is the purpose of annealing in a single PCR cycle?
Allowing primers to bind to complementary sequences
Study Notes
Cloning Vectors
- A popular choice for cloning small foreign DNA is to use a plasmid-based cloning vector, which consists of:
- A cloning site (multiple cloning site or MCS) for inserting foreign DNA
- A selectable marker (e.g. antibiotic resistance gene) for positive selection of transformants
- Elements necessary for propagation and maintenance in the host organism (e.g. a functional origin of replication)
Cloning Vector Components
- Depending on the application, cloning vectors may also contain:
- Elements necessary for expression (e.g. promoter and ribosome binding site, RBS)
- Reporter genes (e.g. LacZα, GFP, GST, Trx) that can be used to form fusion proteins
PCR Amplification
- PCR is widely used in molecular cloning to amplify target DNA from a chosen organism
- For eukaryotic DNA targets, RNA is purified from host cells, converted to cDNA using reverse transcriptase, and then amplified by PCR
PCR Master Mixes
- Convenient ready-to-use formulations of PCR enzymes and reagents that save time and reduce contamination risk
- 2x concentrated solutions containing DNA polymerase, dNTPs, MgCl2, and a suitable reaction buffer
PCR Cycle
- A single PCR cycle consists of three steps:
- Strand separation (denaturation) step: temperature increased to 94 – 98°C for 10 to 30 sec
- Primer binding (annealing) step: temperature reduced to 48 - 68°C for 10 – 40 sec
- Extension step: not mentioned, but implied to be the third step
Molecular Cloning
- A set of experimental methods used to construct recombinant DNA molecules that can be replicated in a suitable host organism
- Techniques used in molecular cloning have advanced rapidly over the past few decades
- Applications include protein expression and purification, recombinant vaccines, and generation of genetically modified organisms
Test your knowledge on molecular cloning, a set of experimental methods used to construct recombinant DNA molecules. Learn about the advancements in cloning techniques over the past few decades, from PCR-restriction-ligation to ligation-free and recombination-based methods.
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