6. Focus: Deciphering a commercial all-in-one kit
Living cells synthesize thousands of different RNA molecules from thousands of genes. However, in the lab, it is possible to synthesize a lot of identical RNA molecules from just one specific gene by conducting transcription in vitro (that is, in a cell-free environment).
Today, many biotechnology companies supply kits that contain all of the necessary components to carry out in vitro transcription. Imagine that you are interested in producing a lot of BST mRNA. (Bovine somatotropin (BST) is a growth-stimulating protein hormone.) You order a $150 kit online and receive a refrigerated box containing some plastic vials and a manual, two pages of which are shown below.
The manual contains a list of components. It immediately bothers you that you have no idea what any of these components really are, so you call ApliGen and ask. They verify that enzyme T7 is indeed present and will carry out transcription. Next, you ask about their UltraNTP mix. The representative on the phone describes it as a mixture of ATP, TTP, CTP, and GTP. You know right then that the kit will not work and ask that they send you a mixture of UTP instead.
The manual contains a procedure for how to conduct the transcription reaction. Before you start, you must first isolate and purify the specific DNA fragment you want to transcribe. The minimal DNA fragment you need to make BST mRNA is the BST promoter. You follow the protocol as described, but the first reaction you set up yields no BST mRNA after an hour. You pick up the phone and call ApliGen to ask for a refund. The representative asks you whether you carried out Step 2 and what your results were. You did carry out Step 2, and you detected a high level of RNA transcripts in the reaction containing pAPLIA DNA.
Which of the following problems could explain your lack of BST mRNA?
...A contaminating RNA-degrading enzyme was present in the "protein-free water."
...Enzyme T7 was stored improperly and denatured before you received it.
...You isolated a BST-encoding DNA fragment that lacked the promoter.
The BST mRNA transcripts produced in your test tube are not like natural BST mRNA because they do not undergo RNA processing. After Step 4, which of the following could you add to your transcripts to cause them to undergo mRNA splicing?
...Nuclear extract from A. thaliana plant cells
....Cytosolic extract from E. coli bacterial cells
...Cytosolic extract from A. thaliana plant cells
...Purified ribosomes and tRNA