The very act of slicing and dicing a piece of DNA with a restriction enzyme was a noteworthy event at one time. Now, even high school students can do it, and much more besides. If you want to bring molecular biology prowess to your lab but don't know what you'll need, read on. Our primer breaks down the essential enzymatic ingredients for starting your own molecular biology laboratory.
The specific enzymes required for any project will depend on what you're doing, and companies such as Ambion, Amersham Biosciences, Fermentas, Invitrogen, New England Biolabs, Novagen, Promega, Qbiogene, Roche Applied Science, Sigma, Stratagene, and Takara offer a vast assortment of options. In many cases, companies bundle reagents necessary for common tasks into kits – a convenient, if somewhat expensive option.
But in any event, you might also consider investing in some lab manuals.
If restriction enzymes are scissors, then DNA ligase is the needle and thread that joins DNA fragments together. Mix two pieces in buffer, add ligase, wait a few hours, and presto! A new plasmid is born.
Integral to any molecular biology experiment, restriction endonucleases are enzymatic scissors that cleave DNA at specific, often palindromic, sequences (e.g., GAATTC). The resulting ends are either "blunt" or "sticky," the latter making for easier cloning operations. Options are nearly limitless: The REBASE database
Polymerases are enzymes that copy nucleic acids. Scientists use DNA polymerases to replicate and label DNA, to make mutants, and to fill in sticky ends, among other things. Check for ancillary activities though: Various pols offer 5'→3' and 3'→5' exonuclease activity as well.
If you want to do PCR, you're going to need a thermostable polymerase. The granddaddy of them all,
No molecular toolkit is complete without an assortment of modifying enzymes. T4 polynucleotide kinase and calf intestinal alkaline phosphatase add and remove, respectively, terminal phosphate groups from DNA. Exo III and mung bean nuclease are exonucleases; they digest nucleic acids progressively from one end. Methylases add a methyl group in a sequence-dependent manner, often negating the activity of corresponding restriction enzymes.
To get a handle on gene expression, you'll need to check the mRNA. As messages are tough to work with, researchers often copy the mRNA into cDNA. The key enzymatic step requires an RNA-directed DNA polymerase, also known as reverse transcriptase (RT). Combine this enzyme with PCR for RT-PCR, and you can amplify specific transcripts.
The hot trend in 2003 was RNAi (RNA interference), a gene-silencing technique. If you require that kind of power, you'll need, at a minimum, a gene-specific siRNA (short interfering RNA) and a transfection reagent to get it into the cell. Alternatively, you can use a specific RNAse – try Dicer or RNAse III – to convert long, double-stranded RNAs into siRNAs.