My professor was gone on a conference today, so this week's Biology 390 seminar was given by our TAs. We went over three concepts which I was already familiar with, but this did present some new facts that I did not previously know. These three concepts were DNA sequencing, Ampicilin resistance, and cDNA libraries.
The method of DNA squencing we will be using in our lab was the method devised by Fred Sanger (which ultimately won him the nobel prize). It utilizes the principal of dideoxynucleotide chain termination. Dideoxynucleotides (ddNTPs) are much like regular dNTPs except for the fact that the are missing the 3' OH (thus making them DIdeoxynucleotides, see picture to the right). Since the 3' OH is necessary for the addition of the following nucleotide during DNA synthesis, if a ddNTP gets incorperated into a growing strand, the synthesis comes to a halt. These are added to a reaction along with template DNA (the DNA that you wish to find the sequence of), regular dNTPs, primers (specific to the regions flanking your template in whatever vector you chose) and DNA Polymerase.
The DNA polymerase used for this method is one that has no ddNTP discrimination, such as AmpliTaq FS - that is to say, it does not incorperate the ddNTPs with any preference. They are added to the growing strand at random. Thus, as one would expect, any reaction would contain multiple strands of varrying lengths - the ddNTPs are added at random, and the strand synthesis halts at random positions. If the reaction is left to run a sufficent amount of time, strands of every possible length are formed. For example, if your template read ACCTG, you would end up with strands that were:
The letters in bold are the incorperated ddNTPs.
Each ddNTP has attached to it two dyes, a donor dye and an acceptor dye. The donor dye, usually fluorescein, when excited by a laser, emits energy which then excites the accetor dye. The acceptor dye, usually rodamine, then fluoresces light of a particular wavelength. The acceptor dye on each specific ddNTP is different and fluoresces at a different wavelenghth - ddATP may have a dye which fluoresces at 565nm, while the dye attached to ddCTP may fluoresce at 615nm. Based on the wavelength emited, one can determine which ddNTP ended a specific fragment.
All the fragments produced by the sequencing reaction are passed through a capiliary in a sequencing machine. As in gel electrophoresis, the larger fragments run through faster than the smaller ones. The capiliary matrix must have a resolution that can discern between fragments differing by as little as one base pair. As the fragments pass through the capiliary, they pass by a laser, which excites the dyes attached to the ddNTPs. The largest fragment passes through first, and the wavelength it emits tells us what the last letter in the sequence is. The next largest is the second to pass through, and from it, we learn what the penultimate letter in the sequence is. This continues until the smallest fragment, representing the first letter in the sequence, is passed through the matrix.
A computer records the wavelengths that are emitted as they pass through, matches them up to their corresponding nucleotide, and arranges the results in the form of a chromatogram (aka electropherogram).
This is a readout of your exact sequence. Sometimes the computer is unable to determine what a particular base might be, and replaces it with an N on the readout. In this case it may be possible to look at the wavelength recorded and determine the identity of that particular base yourself.
This method of sequencing often works for fragments up to 800 bp in size. For fragments longer than this, sequencing in both directions from opposite ends of your fragment is best. For fragments less than 1.6kb, this may give overlapping fragments which can then be aligned to determine the complete sequence. For example, the pBluescript II SK+ plasmid which we are using in our lab contains two promoter sites, a T7 Promoter and a T3 Promoter. These face in opposite directions and flank the region where the template fragment has been inserted. Using these promotors, one can do two sequencing reactions, giving two sequences that can be overlapped at their ends to determine the complete sequence.
The topic of ampicilin resistance was brought up because it is used to select for bacterial colonies which contain a pBS II SK+ plasmid (since the plasmid confers Amp resistance to the bacteria). The gene that does this is refered to as the bla gene, which encodes the enzyme ß-lactamase. This enzyme breaks down the ß-lactam ring found in the structure of Ampicilin and other members of the Penicilin antibiotic family.
Ampicilin itself binds to a class of proteins known as Penicillin Binding Proteins (PBPs) which help in the biosynthesis of peptidoglycan, a component of the bacterial cell wall. By inhibiting the cell wall biosynthesis, the bacteria die. ß-lactamase thus ensures that the cell wall production is not inhibited by Ampicilin.
Unfortunately for molecular biologists, Ampicilin is broken down over time, so plates containing Ampicilin that have been unused for a long time cannot be used to select for Amp resistant strains.
Using Amp resistance is not without its problems. The ß-lactamase enzyme is secreted into the media surrounding resistant colonies. This means that the media immeadetly surrounding the restiant colonies is Amp free. Other non-resistant colonies can grow here. These are known as satellite colonies and will not contain any plasmids, and possibly messing up your experimental results. Another problem is the possible loss of plasmids. Since the media becomes Amp free, there is no selection for colonies that have a plasmid to grow there. Replicating the plasmid before cell division requires energy, and for cells gowing in the Amp free area, it is better for them to not have a plasmid at all. Without selection for keeping the plasmid, it is possible that it could be lost from the population alltogether.
The final topic was that of the creation of cDNA, or Copy DNA, libraries. cDNA libraries are collections of DNA fragments that represent all of the actively transcribed genes in an organism. In other words, for every mRNA in an organism, there is made a DNA "copy" of that mRNA.
First, all the mRNA in a sample is collected. The mature mRNAs in eukaryotes have a poly(A) tail at the end of them after processing. This poly(A) tail acts as a binding site for a poly(T) primer. This primer has three sections: the poly(T) site, a XhoI restriction enzyme site and a run of "GA"s called the "GAGA" site:
The bold portion is the XhoI cutting site.
Next, the enzyme reverse transcriptase is used to transcribe backwards from the poly(T) primer to the 3' end of the mRNA template. This creates a double stranded duplex consisting of one strans mRNA and one strand DNA. This reaction uses a special form of cytosine, 5-methyl-dCTP. When added to the growing duplex, it prevents the cDNA from being chopped up by restriction enzymes.
Next, the RNA template is degraded by RNAse H, and the little pieces of RNA remaining are used as primers for DNA Polymerase I and PFU to replace the RNA strand with a DNA strand. This results in a double strand DNA duplex copied from the original mRNA template.
This cDNA needs to be able to be ligated into a vector, and an additional step is to transform the ends of the cDNA into different sticky ends. Adapters are added to each end which contain an EcoRI cut site. Next, XhoI is added, which cleaves at the XhoI site in the original primer (this is the reason for the 5-methyl-dCTP; the cDNA wont be cut up when XhoI is added). The end result is a cDNA fragment, with a EcoRI sticky end upstream and a XhoI sticky end downstream, allowing for it to be directionally cloned into a vector that has been cut with these enzymes.