Biology 475

Molecular Biology

DNA Sequence Analysis

National Science Foundation

Western Oregon University

Yellowstone National Park

DNA sequence analysis is an intense procedure. Conceptually, the process is amazing; understanding it requires the application of so many central dogma basics. Technically, the process is difficult if you are not patient and manually capable. Cost-wise, the process is not cheap; our lab is furnished with a Li-Cor DNA sequencer (40K), made possible with an NSF-ILI grant and matching university funds. DNA sequencing methods are based on "di-deoxy chain termination methods" developed by Sanger et al. in the late 1970's. In order to understand this method, you first need to review how DNA replication works.

DNA Polymerase requires several things to replicate DNA: (1) single-strand template DNA; (2) a short "primer" complementary to the beginning of the template and providing a 3'-OH group used for addition of new monomers; and (3) nucleotides, the monomers (A, T, G, and C) that will be joined to build the complementary copy. The nucleotides must be in the triphosphate form (called dNTP - for "N" - any ATGC - deoxy-nucleotide triphosphates). In a living cell, nucleotides bear a 5'-Phosphate and a 3'-OH group.

The first thing to understand about sequence analysis is that it works a lot like replication - but is targeted and selective for a few reasons. The first reason is that when you sequence something, you begin with a huge pure population of identical DNA templates... zillions of copies (that's why you spent so much careful time generating large-scale amounts of plasmid). Sequence analysis proceeds AFTER you have a "clone" of a gene; it can NOT be the first step of any research project or fishing expedition for a gene. You also have to know a little something about your template in order to do sequence analysis. The reason for this is because you have to design and build (i.e. buy) specific primers that are complementary to the beginning part of your target. Cloning assists in this process because you have put your target unknown sequence into a vector; thus, you can use known sequences from the vector to "prime" sequence of the unknown insert sequence. After you have read into your insert, you can design new primers (if need be) to "walk" the whole thing.

By beginning with a highly pure population of template and carefully defining your sequencing start point with a manufactured primer, you are programming exactly what you intend to replicate. Getting your primer to stick to your target requires heat to denature the double stranded template. Lowering the temperature slowly - and to a temperature that favors primer binding - will allow the primers to stick.

Now all you need is a DNA Pol and nucleotides to copy. Indeed, mixing DNA Pol and a mixture of all nucleotides would allow replication to fully proceed. The problem is that the product you would get would be fully copied target. And that doesn't tell you anything about the actual sequence. What you need is a way to make a snapshot of every monomer as it is being added. And you do that by using what are called di-deoxynucleotides (ddNTPs). ddNTPs are called chain terminators because they lack a 3'-OH group. If one ddNTP is added to a growing chain, that complementary chain will end right there.

Nucleotide sequence analysis works because it uses a MIXTURE of dNTPs and ddNTPs (less of the latter). There is a "race" for each position by normal dNTPs and chain-terminators. Think about this (and let your mind expand a lot because it is a HUGE concept): you start with a zillion templates. That means you can - by chance alone - create stop products that represent EVERY single potential stop product (even if you are, like, sequencing a gene that is thousands of base pairs long - WOW). Once you accept this, you have to step back again and think about the physical logistics of separating these products and seeing them. First, the separating part: a special gel made from polyacrylamide is capable of "resolving" DNAs that differ by ONE NUCLEOTIDE. The principle of separation is the same as in agarose gel electrophorsis - only the chemicals that build the gel (both their resolving power and their toxicity) are different.

Second, the labeling part. Our manufactured primers have each been tagged with fluorescent compounds. Because each template must have a primer, we know that every product will have label. To make things even sweeter, the sequencing machine has a "reader" that scans back and forth across the gel as it is running (some ungodly number of times per minute) and "captures" the image to a computer screen. It actually works just like a spectrophotomer, capturing the specific wavelength emitted by the fluorescent tag. The last thing that has to be emphasized (and this will require, perhaps, some thought and drawing on your part) is that you cannot put all four chain terminators in the same tube. If you ran this out, you would get a continuous ladder of information and you would not be able to figure out which product ended with which nucleotide chain terminator. So - what you actually do is set up FOUR reaction tubes:

    (a) all four dNTPs plus only ddATP
    (b) all dNTPs plus only ddGTP
    (c) all dNPTs plus only ddTTP
    (4) all dNTPs plus only ddCTP

When you run out these products, you run each in a separate "lane" of the gel. Then you read the bands and KNOW which chain terminator they end with and, hence, the sequence. Got all that?

Reaction Set-Up Procedures

As recommended by Li-Cor, the maker of our DNA sequencer, we utilize the Epicenter thermal cycle sequencing kit. This kit is NOT cheap. 100 reactions cost 200 dollars.

This protocol is based on what I call the "rainbow" Epicenter Kit and contains the following reagents:

    (1) Taq Pol: thermal stable polymerase
    (2) Buffer
    (3) Red Stop G Mix: termination G's plus all dNTPs
    (4) Yellow Stop T Mix: termination T's plus all dNTPs
    (5) Green Stop A Mix: termination A's plus all dNTPs
    (6) Blue Stop C Mix: termination C's plus all dNTPs
    (7) Loading Dye

You will supply your template of interest and a fluorescent-labeled primer specific to your template through the following procedures.

Set up four joined mini-microfuge tubes and one orange cocktail tube. The orange tubes are for the "pre-mix" cocktails (primer, template, buffer, and polymerase) and the yellow, red, blue, and green tubes correspond to each of the color-coordinated ddNTPs. Label by writing the clone number on the tube lid in VWR pen ink. Do not worry about closing the tubes now or anytime between additions. There is not enough time to do this. Time - and keeping things on ice - is of the essence.

In orange tubes, mix the following well (but make sure everything is at the bottom by the end):

    (a) Template (7.3 ul plasmid, which corresponds to recommended 3-10 ug)
    (b) Flourescent-labeled primer (1.5 ul)
    (c) Buffer (7.2 ul)
    (d) Thermal Stable Polymerase (1 ul)

Add 2.0 of the appropriate ddNTP stop mix to each color-coordinated tube. Physically place in the bottom of the tube.

Now, CAREFULLY, add 4.0 of the primer/template to each of the four termination mix tubes. After experimenting, we found that we could simply add each to the side of the tube (about mid-way down), moving quickly down the line with the same tip per individual template. Change tips BETWEEN SAMPLES!

Add 6 ul of oil to each tube; you can do this using a single tip as long as you add drop to UPPER side of tube and have been diligent in the prior steps.

Flick each tube to mix, pulse to down quickly, and place on ice.

Carry to the thermal cycler and carry out program 47, which takes approximately 90 minutes. Program 47, already programmed in, performs the following steps that you should understand in terms of purpose:

    (1) Denaturing I: 92°C for 2 minutes
    (2) Denaturing II: 92°C for 30 seconds
    (3) Annealing: 50°C for 15 seconds
    (4) Extending: 70°C for 15 seconds
            * Repeat 2-4 for a total of 30 cycles. End with a 4°C soak.

Gel Set-Up Procedures

Pouring, setting up, and loading a sequencing gel are complicated, tedious, and fussy skills that take tons of experience to do well. I have tried to summarize general ideas here but NOTHING - I repeat - NOTHING will substitute for doing it over and over again. This protocol is based the Li-Cor sequencer we have; many general concepts are transferable to other sequencing systems. These methods are to be carried out WHILE the sequencing reactions are polymerizing. If done correctly, you will actually make perfect time. If you screw anything up, you could be set back at least an hour.

Preparing Your Plates

Thoroughly wash plates as directed. Remember that these cost 100 or more a pop; DO NOT BREAK THEM. Use special detergent and brush provided; emphasize the faces with the notches using diagrams from Li-Cor.

Rinse first with tapwater thoroughly; then rinse with distilled water in the carboy by the sink as your FINAL step.

Set in the drying rack while you work in the next several steps.

Preparing Your Catalysts and Acrylamide Solution

All equipment related to sequencing is dedicated to my back sequencing lab. Do not leave it in common areas after use. Gel reagents are messy and salt; wash everything immediately when through!

In contrast with agarose gels, polyacrylamide gels require two catalysts to harden.

One is APS, which must be mixed FRESH each time. Weigh 0.05 g using the ultra-fine scale in the upstairs chemical stock area. Place in a microfuge tube. fill to 0.5 ml with ddwater. Label and set near near the acrylamide mix (but not on ice).

The other is TEMED. Obtain TEMED from the refrigerator in the microbiology preparation room; keep on ice during use. Remember to put this back when you are finished.

For the actual gel, measure 17 grams Urea into a dedicated 150 ml beaker and BARELY cover with distilled water. Total volume cannot be greater than 30 ml or the rest of the ingredients won't fit and your measurements will be off. Add a stirbar and heat until in solution. During heating, you may need to swirl it to bring down any crystals on the edge.

Bring the urea solution back to the lab and add it to the dedicated 50 ml graduated cylinder. To this, using respectively dedicated pipettes, add 4 ml 10X TBE and 4.4 ml Long Ranger acrylamide solution. Remember that acrylamide is a neurotoxin (albeit - it is most dangerous in an inhaled powder). Bring up the volume to 40. If anything comes out of solution, you will have to reheat.

Pour into a new clean 150 ml beaker. Place mix on ice. Mixture MUST be on ice or it will polymerize too fast in subsequent steps. Make sure you have a 60 ml syringe clean and dry. Likewise, make sure you have a "green syringe tip" clean, dry, and ready to go.

Assembling and Pouring the Gel

Place non-eared gel down with notch up. Using only chimwipes, dry/buff glass face with 95% ethanol until bone-dry.

Place 0.2 mm spacers along the sides; dot with tiny speck of water to make them adhere.

Wipe down notched face of eared gel. Place on spacers on non-eared gel plate (notches should both be at the bottom, meeting).

Clamp as directed with the black screw-clamps. Not beyond "tight" for wimpy me. Got that Duuuuuudes.

Place entire set-up at the set "angle" in the holder. Make sure you have the comb and the comb-press clean, dry, and ready.

The next several steps are high stress; use the bathroom and do whatever you have to do now.

Add your catalysts: 270 ul APS solution (swirl gently after adding); 27 ul TEMED (swirl gently again).

Now, you have about 5 minutes.

Using the clean tip-less syringe, suck up all but about 5 ml acrylamide.

Place a green wide-bore tip on the end while holding the full syringe upside-down.

Load by injecting into the top of the gel. Inject forcibly but evenly. Your team-mate should be standing by with the "bubble-grabber," ready to assist. The bubble-grabber is a thin platinum wire that can be slid between the plates to remove bubbles (within reason). It is not as easy as it looks. It's also hopeless within five inches of any edge. Have the assistant dot (with an alcohol-based marker) any bubbles you cannot remove for future reference.

After the acrylamide solution has descended, filling the space between the plates, lower the gel to a flat position, place the comb in "upside down" (flat side in all the way). Place the comb press on the comb and secure.

Eject the remaining acrylamide from the syringe into the original beaker and place next to gel (i.e. not on ice). This will "tell you" whether polymerization happened.

Within 20 minutes, the beaker of waste acrylamide should be solid. The gel still has to "cure" a full hour. Go have a snack after you've washed your hands.

Gel Set-Up

Mix 1000 ml 1X TBE using the 10X lab concentrate; if you can't do this in your head, you need to practice this stuff.

After curing, place gel vertically in holder, remove comb press, and clean all gel bits/crystals from notches around comb. Do not remove the comb until this is done. Spray with a lot of distilled water!

When satisfied, carefully remove comb and clean away any grit that was behind the comb. Make CERTAIN you can visualize the long "well" left by the comb. Clean out using TBA sprayed from a syringe with a blue narrow-bore tip.

When confident the well is clean, gently place comb in at first (i.e. slide in about 1 mm, making sure ALL teeth are between plates and not getting crunched). When even, firmly and decisively place fully into the acrylamide front. The teeth should actively poke into the gel line 1 mm - no more!!! Make sure I am around for this step because losing it here really sucks.

Assemble upper buffer chamber as directed and using gasket.

Place lower buffer chamber on sequencing apparatus and fill minimally with 1X TBE (do not splash anywhere without wiping down thoroughly).

Clean and dry/buff plate faces (lots of water plus final 95% ethanol step). Gently set the whole thing onto sequencing machine, being very careful not to slosh any liquid down the back.

Fill upper buffer chamber and put appropriate covers on upper and lower buffer chambers. The lower chamber cover serves as the electrode connection. The upper electrode, however, must be added. Do so.

Turn on sequencing computer and set up as directed (this includes naming and saving project files, focusing AND auto-gaining). These steps will take a fair bit of time.

Pre-run 5-10 minutes, until 50 degree temperature is reached.

While pre-running, carry out all final reaction steps.

Reaction Finish and Load

Set up a boiling water bath.

Gather your sequencing tubes from the PCR machine.

Add 3 ul red/magenta loading dye to the upper side of each; pulse-centrifuge to spin down.

Boil tubes for 3 minutes. During this time, turn off gel pre-run and spray out each well for a second or two with TBE using blue tip/syringe. I call this "faith cleaning" because I don't really watch - I just go down the line.

Following boiling, load 1.5-2.0 ul of each reaction with fan-tip (wipe oil before each on a paper towel). I'm not about to describe this; you either see it and can do it or you can't and need more practice. Make sure your team-mate is writing down what you load as you load it. Call out colors and numbers and report any spaces, problems, etc. These should all be recorded in your notebook.

Run overnight; clean up the next day.

Freeze remaining reactions; they are good for one week.