Genetics 3250 Sequence Analysis
Cahoon – Genetics 3250 Lab
Middle Tennessee State University
Chloroplast Genome Project – Week 2
DNA Sequencing
Goal – sequence portions of our inserts.
Materials
· Template DNA (the cloned pieces of DNA in plasmid vectors)
· Primer (T7 and/or T3)
· Sequencing Reaction mix (Beckman-Coulter Master Mix)
· Beckman-Coulter Genetic Analyzer automated DNA sequencer.
Introduction
The dideoxy termination method for sequencing DNA was developed in the 1970’s. It takes advantage of the natural process of DNA replication coupled with modified nucleotides that halt synthesis at specific places on a DNA template. In this process each piece of DNA that is produced is permanently stopped at a specific nucleotide. Over the course of multiple reactions a complete set of nested products is produced. Each product differs in length from its counterparts by as little as a single nucleotide. The products are distinguished and visualized through separation by electrophoresis and visualization by either radioactive isotopes or fluorescent tags.
The original method was simple enough to be performed in most molecular biology labs but was labor intensive. It wasn’t until the advent of automated DNA sequencing that large genome sequencing was realized. We will use an automated DNA sequencer that uses dideoxy termination chemistry and modified termination nucleotides with attached fluorescent dyes. The dyes are excited by a solid state laser and emitted light is detected by a spectrophotometer within the sequencer. The light signature is interpreted by a probability program which assigns a base (A,G,C, or T) to each fragment.
The sequencing reaction
If we want to use DNA replication
for sequencing we have to satisfy all
of the criteria needed for that process.
1. Double stranded DNA must be
denatured to allow a primer to anneal
2. DNA synthesis can only begin
after a primer is in place.
3. Once a primer is in place the
sequencing reaction can begin.
DNA Polymerase will add bases
which complement to the template.
Mostof the nucleotides provided
for the polymerase are normal bases
(represented as A, T, G, and C in
the figure) that allow typical
polymerization.
4. This will continue until one of the
modified “terminator” bases (represented
as A**, G**, T**, C**) is added.
This stops synthesis of the strand. In
addition, each terminator base carries
a unique fluorescent marker which allows
us to determine exactly which base it is.
This series of figures shows one
incidence of this type of reaction. We have
to imagine now that this reaction is taking place simultaneously on thousands of templates. Each reaction results in a product that ends at a different base. If enough reactions are run then every single base should be represented in our products.
5. Our reaction products are now separated by size using a technique called gel electrophoresis. Electrophoresis will occur within the sequencer.
Within the sequencer the fragments are
separated by size. As each piece of DNA
passes the laser, the dye on the end fluoresces
and emits light at specific wavelengths.
A spectrophotometer detects the light.
A computer program then interprets the light
signature and assigns a base.
There are four unique tags (one for each
nucleotide) which emit four different
fluorescent patterns.
Where do the primers come from?
You might be wondering how primers (which must be a known sequence) were designed to recognize a portion of a genome that has not been sequenced. There are several ways to do this. Since this is a class for beginning geneticists I will limit this discussion to the approach we will employ. Briefly, Dr. Cahoon compared the sequences from three grass chloroplast genomes (maize, rice, and wheat) by aligning similar regions like those shown below. He then looked for regions that were the same in all three genomes like in BOX 1. He then made the assumption that these regions may also be conserved in other grass species and designed primers that would anneal to these regions. BOX 2 demonstrates a region that is not conserved between the three.
This is a modern “post-genomics” strategy. Meaning, we are using existing data to facilitate sequencing unknowns. As recently as five years ago this strategy would not have been possible
Manipulating and Working with DNA
When working with DNA we have to treat it like any other component in a chemical reaction. This means it is necessary to know the concentration. Our first exercise is to quantify DNA using spectrophotometry.
DNA exercise 1. Quantify your plasmid DNA solution using a spectrophotometer!
A spectrophotometer can shine light of a specific wavelength through a solution and then record how much light is absorbed by the solution.
DNA will absorb light at 260nm (ultraviolet range).
a. Make a 1/100 dilution of the DNA you isolated last week by adding 5 microliters of DNA to 195 microliters of water.
b. Put your dilution into a quartz cuvette and read the absorbance at 260nm.
An absorbance unit of 1 at 260nm (A260=1.0) = 50 ng/ml double stranded DNA.
c. Use the formula below to calculate the density of your DNA.
______X 100 X 50ng/ml = ______ng/ul
(A260) (dilution)
d. If you needed to add 500ng of DNA to a reaction how many microliters of your solution would you add?
500ng / ______= ______ul of your DNA
(your concentration)
DNA exercise 2. Set up a sequencing reaction.
Preparing enzymatic reactions from “scratch” is relatively easy once you get used to handling the tools and components. But since it is very likely that this is the first one you’ve ever attempted it has been simplified a bit. This is to ease everyone into using things like micropipettes and to maximize the likelihood of success – few things in research are as disappointing as carefully planning a reaction and getting nothing out of it. Several components have been pre-measured and mixed. At each step the pre-mixed components are described. If you want to learn more you may try taking our Biotech class or attempting an independent study in a molecular biology lab.
1. The DNA has been quantified and appropriately diluted. Add 10ul of the DNA mix to a reaction tube containing 8 microliters of reaction master mix.
The master mix contains pre-mixed…
DNA polymerase
Normal dideoxy nucleotides (A,T,G, and C)
Labeled terminator dideoxy nucleotides (A**,T**,G**, and C**)
Reaction buffer
Which piece of the chloroplast genome did you add to your reaction mix?
______
2 Add 10pmoles of primer to the reaction tube by adding 1μl of the pre-mixed primer.
Which primer did you add?
Your instructor has a “primer key”. Find your primer name and write the sequence below.
3. The sequencing reactions will be run and loaded into an automated sequencer in the biotech lab (room 131 DSB) by your instructor. These remaining steps will take about 7 hrs to complete. Your sequence will be available next week.
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