Our overall strategy is called "shotgun" sequencing. As a first step, we break the starting DNA randomly into pieces of about 1 kb. These pieces are inserted into an M13 vector and sequenced. Since these pieces are random, we can use the overlapping sequences to assemble back to the starting DNA, which can be a cosmid, a recombinant lambda DNA, a DNA band isolated from a gel, total genome, etc. Explicitly, we employ a "linker-adaptor" methodology to avoid constructing unwanted chimeras during the ligation of insert to vector.
For purposes of presenting these methods, we first describe the vector preparation and, then, describe the insert preparation. It is truly vital to have an excellent vector in order to produce a sequencing library. We are employing the male-spe cific coliphage M13 as the sequencing vector. M13 is a filamentous phage with a single-stranded, circular genome. M13 is widely used as a vector because many versions are available commercially and because M13 has certain advantages: a) the filamentous phage coat easily increases in size to accomodate larger DNA; b) as the DNA is already single-stranded, no denaturation is required before the se quencing reactions, and there is no competing renaturation reaction; c) there is an in-phase polylinker sequence behind an inducible lac promoter, yielding blue or white plaques; and d) a universal primer, upstream of the polylinker sequence, is available commercially - even four different dyes are available commercially as dye -primers.
M13 VECTOR PREPARATION
1-For cloning in M13, the starting material is the covalently closed duplex, circular replicative form (RF) of M13 DNA. We have frozen stocks of M13-infected E.coli NM522. (E. coli NM522 is male, grows M13 well, and is available commercially.) A standard 3-way streak is made on an LB plate, and the plate is incubated overnight at 37°C. First thing in the morning, an individual colony of M13-infected E. coli NM522 is placed in 5 ml of LB media. The tube is placed on the 37°C wheel all day. Late in the day, the turbid 5-ml culture is used to inoculate 500 ml of LB (in a 2-l flask). The flask is placed in the 37°C incubator/shaker overnight at 250 rpm. The culture is harvested the next morning.
2-M13 RF DNA is effectively a high-copy number plasmid and can be isolated as such. We employ a Qiagen Midi-prep kit. At the first step, E. coli (containing M13 RF DNA) is pelleted by centrifugation. The supernatant contains a high concentra tion (approximately 5x1011 pfu/ml) of M13 phage. If you want M13 single -stranded DNA, you can harvest the phage in the supernatant.
3-The final step in the Qiagen Midi-prep procedure is to precipitate the M13 RF DNA by the addition of isopropanol. To accomplish this step, we collect the final effluent (containing the M13 RF DNA) from the column in a 15-ml Corex tube. The isopropanol is added; the tube is covered tightly with parafilm, and the contents mixed. The parafilm is carefully removed. The tube is centrifuged in an SS34 rotor plus adaptors in a Sorvall RC-5B centrifuge (or equivalent) at 12,000 rpm for 30 min at 10°C. The supernatant is poured off gently, so as not to disturb the pre cipitate. 10 ml of cold 70% ethanol are added gently, again so as not to disturb the precipitate. The tube is spun at 12,000 rpm for 10 min at 10°C. The super natant is again poured off gently. 10 ml of cold 95% ethanol are added. The Corex tube is spun for a final time at 12,000 rpm for 10 min at 10°C. The super natant is poured off gently. (Note that the DNA precipitate may not be visible.) The Corex tube is drained briefly and air dried.
4-The precipitated M13 RF DNA is probably not at the bottom of the Corex tube (because the rotor is at an angle, rather than parallel, to the force lines.) The DNA is (probably) spread on the distal inner surface of the Corex tube. 2.5 ml of TE are added. Be sure to wash down the sides of the tube.
5-These DNA preparations contain traces of contaminanting RNA and protein. Add 0.1 ml of RNAse (1mg/ml; DNAse-free). Incubate at 37°C for 1 hr to remove contaminating RNA. Add 0.1 ml of proteinase K (1 mg/ml) and incubate at 37°C for 1 hr to digest contaminating protein (including RNAse and proteinase K itself).
6-Add an equal volume (2.7 ml) of phenol: chloroform (1:1). Cover the Corex tube tightly with parafilm and vortex hard for 10 sec. Remove the parafilm carefully, and centrifuge at 10,000 rpm for 10 min at 10°C (Sorvall RC-5B plus SS34 rotor, or equivalent). Decant the upper, aqueous phase to a fresh 15-ml Corex tube. (Don't be greedy and try to remove all the upper phase. Protein is at the inter face.) Precipitate the M13 RF DNA by the addition of 2 volumes of cold ethanol. Centrifuge the Corex tube for 30 min at 12,000 rpm at 10°C. Gently decant the supernatant, so as not to disturb the precipitated DNA (which is usually invisible). Gently add 10 ml of cold 70% ethanol, without disturbing the precipitate, and centrifuge for 10 min at 12,000 rpm at 10°C. Gently decant the supernatant. Gently add 10 ml of cold 95% ethanol, and centrifuge at 12,000 rpm for 10 min at 10°C. Gently decant the supernatant. Drain the tube briefly and air dry. The precipitated M13 RF DNA is not usually visible but is on the distal inner side of the tube. Add 1 ml of TE to dissolve the precipitate. Be sure to wash down the sides of the tube. The concentration of the DNA can be determined by reading the absorbance at 260 nm in a UV spectrophotometer: at A 260 = 1, M13 RF DNA concentration = 45 µg/ml.
7-Digest a large amount of M13 RF DNA with HindIII. It is important that the diges tion is more than 99% efficient, otherwise blue plaques will appear later (see be low). To achieve efficient HindIII digestion, use the optimal buffer, and add the restriction enzyme in increments over several hours. HindIII recognizes the duplex
sequence 5'AAGCTT 3' and makes staggered cuts between the two A's. The product is a full-length linear duplex DNA of size 7.1 kb with 4-base single-strand extensions at each end: 5'AGCT. Short single-strand extensions are highly vulner able to random nuclease attack. Great care must be taken to keep those 4-base extensions intact. In particular, these DNAs should be stored at -70°C.
8-Dilute the HindIII-cleavage reaction with TE to a final volume of 400 µl. Add an equal volume (400 µl) of phenol: chloroform. Vortex hard for 10 sec. Microfuge at maximum speed for 5 min. Using P200 (or equivalent), decant the upper, aque ous phase to a fresh 1.5-ml eppendorf tube. Add 2 volumes of cold 95% ethanol and mix to precipitate the HindIII-cleaved M13 RF DNA. Microfuge at maximum speed for 5 min at 4°C (if possible). Gently pour off the supernatant, leaving the precipitate undisturbed. (The precipitated DNA may or may not be visible, depend ing on the amount.) Gently add 1.2 ml of cold 70% ethanol. Microfuge at maxi mum speed for 5 min at 4°C (if possible). Gently decant the supernatant and air dry. Dissolve in 200 µl (or other appropriate volumes) of 0.01 M Tris, pH 7.5. Store frozen at -70°C. We do not use EDTA in the storage buffer because EDTA will inhibit the next (ligation) step.
9-The concentration of the HindIII-cleaved M13 RF DNA is determined by measuring the absorbance at 260 nm (against 0.01 M Tris, pH 7.5, buffer) using a UV spec trophotometer. For a duplex DNA, an A260 = 1 is equivalent to 45 µg/ml, mea sured as bases or phosphates per ml. We need to know the concentration in terms of M13 RF DNA molecules per ml. (Technically, we need to know the concentration of HindIII-ends per ml, which is simply twice the concentration of DNA molecules.) Use the molecular weight of M13 RF DNA (7.12 kb = 7.12x10 3 base pairs times 660 mol weight per base pair yields a mol weight of 4.7x10 6 for M13 RF DNA) to convert the concentration to units of number of DNA molecules per ml.
10-Separately, synthesize or purchase the phosphorylated 9-mer, 5'pAGCTGTTTG3', hereafter called the "linker". The first four bases of the linker, 5'AGCT, are complementary (in the Watson-Crick sense), when anti-parallel, to a HindIII end. We are going to ligate the linker to the HindIII-cleaved M13 RF DNA. To achieve that ligation, the linker must be phosphorylated on the 5' end, as the ligase cannot add a phosphate. The ATP in the ligation reaction provides energy, not a donor phosphate.
11-The usual final step in oligomer preparation is to dry the oligomers (e.g. Speedvac). Dissolve the 5' phosphorylated 9-mer linker in a minimum volume of 0.01 M Tris, pH 7.5. We want a highly concentrated solution. We do not use EDTA in the buffer, as EDTA inhibits the ligase reaction. Aliquot and freeze ( -70°C) the linker.
12-Determine the concentration of the linker by measuring the absorbance at 260 nm (against 0.01 M Tris, pH 7.5) using a UV spectrophotometer. For a short single-stranded oligomer, an A260 = 1 is equivalent to 35 µg/ml, measured as bases or phosphates per ml. We need the linker concentration in units of mol
ecules per ml. Use the molecular weight of the linker (9x330 = 2970) to convert the linker concentration to molecules per ml.
13-Now that you have the HindIII-cleaved M13 RF DNA expressed as concentration of HindIII-ends per ml and the linker concentration expressed as molecules per ml, you can calculate the ratio of linkers to HindIII ends. On the basis of this calcula tion, you may ligate linker to HindIII-ends in the presence of a 100-1000 molar excess of linker: that is, the number of molecules ratio, linker : ends = 100-1000. However, the measurement and calculation of linker concentration assumed that the linker preparation was 100% pure, and that is not always the case. The phos phorylated linker may contain, as an impurity, the 9-mer without phosphorylation. In addition, prematurely truncated oligomers (e.g., 8-mers) may be present as impurities.
14-We employ a functional assay to measure effective linker concentration. If you have HPLC-purified linker, you may wish to assume that it is reasonably pure. On the other hand, the preparation of excellent vector is vital to the whole sequencing effort. For our functional assay, we employ HindIII-cleaved lambda DNA: mol weight = 3.2x107. Complete HindIII cleavage of lambda DNA produces 14 HindIII -ends. From the spectrophotometrically-determined concentration of lambda DNA, the concentration of HindIII-ends can be calculated. Alternatively, you can pur chase lambda DNA cleaved by HindIII.
15-When ligase (T4 DNA ligase), buffer, and ATP are added to HindIII-cleaved lambda DNA, a high molecular weight DNA is constructed. This DNA migrates very slowly in an agarose gel. If linker is added to the ligation mix, the linker competes for the HindIII-ends. When ligated to a HindIII-end, a linker prevents further ligation and prevents the formation of high molecular weight DNA. We titrate linker into the ligation mix and look for the amount of linker necessary to completely block the formation of high molecular weight DNA. We calculate the ratio of linker : HindIII ends at complete blockage based upon the nominal concentration (that is, assuming 100% purity) of linker and the accurrate concentration of lambda HindIII -ends. With different preparations of linker, this value has ranged between 100 to 1000. The ligation reaction is bimolecular, whereas the reaction of linear HindIII -cleaved M13 RF DNA circularizing is monomolecular. Thus, the two reactions are not the same. Nevertheless, our measurements of the functional linker concentra tion have proven very useful.
16-Heat HindIII-cleaved M13 RF DNA to 60 °C for 10 min, and then cool on ice for 10 min. Add linker (at the functional ratio for blocking ligase), buffer, ATP, and T4 DNA ligase. Incubate at 16 °C for 6-8 hr (some prefer to incubate at 16 °C over night). The temperature, 16 °C, is chosen to be below the melting temperature of the 4 base pairs we want to form. At the end of the incuabtion, either freeze the sample -70°C, or take it directly into an agarose gel (1% agarose in 0.5X TBE).
17-Purify the vector (linker ligated to HindIII-cleaved M13 RF DNA) by agarose gel electrophoresis. Excess linker migrates well ahead of vector (7.1 kb). Unwanted,
re-formed circular M13 RF DNA (formed by ligation of HindIII-cleaved M13 RF DNA to itself) migrates as if it were (approximately) 15 kb, behind vector. Very small amounts of higher order forms migrate even more slowly. We run the gel long enough to run the excess linker off the gel, and get better separation of the vector and higher forms.
18-Vector is now eluted from the gel. There are many methods for eluting DNA from agarose, including electroelution, hot phenol, Geneclean, Nucleotrap, etc. At present, we are using Qiaquick (from Qiagen), because it is fast and simple. To protect the 5 base single-strand extensions, speed counts. We elute the vector from the Qiaquick columns with 0.01 M Tris, pH 7.5, and freeze (-70°C) in aliquots. Self-ligated vector should yield no plaques.
DNA Insert Preparation
After preparation of the vector, the second part of the sequencing library con struction is preparation of the DNA insert. The following procedures have been optimized for the Olson cosmids and recombinant lambda DNAs that comprise yeast (S. cerevisiae) chromosomes IV and V. These various yeast recombinant DNAs can be produced only in small amounts. Thus, our first priority during insert preparation is conservation of the recombinant DNA.
To prepare insert DNA, firstly, the cosmid (or lambda DNA, etc.) is broken ran domly into pieces of approximately 1 kb in size. Secondly, enzymatic procedures are employed to prepare ("adapt") the DNA for ligation into the M13 vector.
Random Size Reduction
Randomness during DNA size reduction is an essential feature of this experimental design (see Assembly). Historically, sonication (i.e., breaking the DNA by cavitation) has been employed to acheive DNA size reduction. Unfortunately, sonication conditions are difficult to control and very difficult to reproduce. We strongly recommend against sonication and strongly recommend in favor of shear breakage (i.e., breaking the DNA by stretching it).
Peter Oefner, in our laboratory, devised our DNA shearing procedure. Peter uses an HPLC apparatus: a pump capable of producing high pressure and the tubing, of different inner diameters, able to withstand the pressure. Scott Smith, in our laboratory, is building an inexpensive stand-alone shearing apparatus, so that an expensive HPLC need not be purchased. An actual protocol awaits the stand-alone device.
Blunt-ending the DNA
Regardless of the method that we use to reduce DNA size, we need the physical ends of the DNA to be flush: i.e., a correct base pair, with 5' phosphate and 3' hydroxyl. Other types of ends are of no use to us. Some otherwise useless molecules, those with 5' single-strand extensions, can be converted to useful DNA by the action of DNA polymerase. (Other types of useless molecules are not affected.)
In jargon terms, we convert "jagged" ends to "blunt" ends, or we "blunt-end" the DNA, or we "fill-in" the ends. For this purpose, we use a DNA polymerase with little or no exonuclease activity: either the Klenow fragment of E. coli DNA-dependent DNA polymerase I or T4 DNA-dependent DNA polymerase. During the polymeriza tion reaction, we do not want to synthesize a lot of DNA; we want only to fill-in the otherwise single-stranded ends. We restrict the reaction by carrying it out at 11°C and for only 1 hr. Dilute DNA polymerase is unstable. We add acetylated-bovine serum albumin (BSA) as a protein carrier to stabilize the enzyme. The acetylation is not actually directed at the BSA. Rather, it is meant to remove any extraneous enzymatic activity (impurity) by acetylation.
1-Set the microcooler to 11.0 °C. It will take at least 30 min to cool down from room temperature. Thaw and assemble the reagents on ice:
Pre-cool the eppendorf tube on ice. All these reagents are kept on ice so that the DNA polymerization will not start prematurely. Mix the following reagents: (a rough guide is given here)
Note: it is important to dilute the EDTA with the DNA (stored in TE) and the glycerol with the enzyme.
2-Incubate at 11 °C for 60 min.
3-Following the DNA polymerase reaction, the DNA must be re-isolated in pure form. The efficiencies of DNA purification methods are a strong function of the amount of DNA. Therefore, we recommend using one of two procedures, depending on the amount of DNA. These two methods are only a guide. If you have a method you like, use it.
4-If you have a lot of DNA (more than 20 µg), use TE to increase the reaction volume to 400 µl. Add an equal volume (400 µl) of phenol : chloroform (1: 1, with the phenol previously equilibrated with TE) and vortex hard for 10 sec. Microfuge at maximum speed for 5 min. Using a P200, gently decant the upper, aqueous phase to a clean eppendorf tube. The protein is at the interface and in the organic phase. Don't be greedy and decant too close to the interface. Decant approxi mately 300 µl. To precipitate the DNA, add 2 x volume of (-20 °C) 95% EtOH. Vortex hard for 10 sec. Microfuge at max speed for 5 min. Gently, so as not to disturb the (usually invisible) DNA precipitate, pour off the supernatant. Gently add 1 ml of cold (-20 °C) 70% EtOH. Microfuge for 5 min. Gently pour off supernatant.Gently add 1 ml of cold (-20 °C) 95% EtOH. Microfuge for 5 min. Gently pour off supernatant. Drain briefly and air dry briefly. Dissolve the DNA in 50-100 µl of 0.01 M Tris, pH 8.0. Store frozen; freezing is especially important because there is no EDTA in the buffer.
5-On the other hand, if (as is our usual case with the Olson recombinant DNAs) you have very littleDNA ( 10 µg) do not purify the blunt-ended DNA by extraction with phenol: chloroform. You will lose too much DNA. There are several commer cial products (e.g., Nucleotrap, Geneclean, etc .) that absorb DNA onto a matrix. After high-salt washes, the DNA is eluted in low-salt buffer. We employ the Nucleotrap from Macherey-Nagel because the procedure does not require heating, is buffer insensitive, and does not use glass or NaI. We follow the manufacturer's instructions, using only room temperature incubation. In the final step, we elute the DNA with 20 µl of 0.01 M Tris, pH 8.0, and store the DNA frozen at -70 °C (which is very important in the absence of EDTA).
Ligate adaptors to inserts
To construct a recombinant DNA library using the "linker-adaptor" strategy, we must add adaptors to the DNA. Our adaptor was designed by Fred Dietrich of our laboratory (Fred's adaptors = FRADP).
1-Synthesize or purchase the following two oligonucleotides:
FRADP-W 5' pTGAGTCACCAAAC 3'
(13-mer)
FRADP-C 5' pGTGACTCA 3'.
(8-mer)
These oligonucleotides must be phosphorylated on the 5'-strand. The W and C strands are complementary when antiparallel, forming 8 base pairs and having a five base single-stranded extension.
5' pTGAGTCACCAAAC 3'
3' ACTCAGTGp 5'
These five bases are homologous to the linker and are not self-complementary.
2-To prepare the adaptor, dissolve the oligonucleotides in a minimal volume (e.g., 11 µl) of 0.01 M Tris, pH 7.5. (Store the oligonucleotides at -70 °C). Determine the concentration of each oligonucleotide by reading the absorbance at 260 nm and using the conversion factor that, for a short oligonucleotide, an absorbance of 1 at 260 nm is equivalent to 35 µg/ml. This concentration unit is in µg of bases or phosphates per ml. We need to convert the units to µmoles or number of mol ecules per ml. As an example, we can convert µg/ml = 10 -3 µg/µl = 10-3 g/l. FRADP-W is a 13-mer, and its molecular weight is 3996. Thus, the molar concen tration of FRADP-W is the weight concentration, expressed as G/L, divided by the molecular weight (3996) of the molecule. The molecular weight of the 8-mer, FRADP-C, is 2483. We want to combine the two oligonucleotides in as high a concentration as possible (e.g., each 100 µM ) and in a 1: 1 molar ratio.
3-Mix a 1: 1 molar ratio of FRADP-W and FRADP-C at high concentration. To an neal the two strands, bring the solution to 50 °C for 10 min. Remove the tube from the water bath, and allow the tube to cool to room temperature. Lastly, place the tube in a 16 °C bath overnight. The melting temperature of the 8 base pairs is below room temperature. Therfore, do not allow the annealed adaptor to warm up to room temperature, but always keep it on ice.
4-From the 16 °C bath, aliquot the adaptor into iced eppendorf tubes, and freeze the aliquots at -70 °C.
5-Thaw the blunt-ended DNA (20 µl), and place the tube on ice. Thaw an aliquot of adaptor on ice. All reagents are on ice. Add the reagents in the following order to the DNA on ice:
for small amounts of insert DNA,
for larger amounts of DNA:
The ligase is the expensive, high concentration reagent, because ligating blunt ends together is a relatively low efficiency event. We work at adaptor excess. The DNA with adaptor is in buffer that does not contain EDTA. If either DNA or adaptor were dissolved in TE, the EDTA would reduce adversely the amount of Mg +2 present in the 5X buffer. Incubate the tube for 6-8 hr at 16°C.
In the preceding blunt end ligation, there were two species of DNA with blunt ends: the recombinant DNA fragments, and the adaptors. Adaptor dimers form. These adaptor dimers and free adaptors must be separated from the fragments onto which adaptors were ligated. On a 1.0% agarose gel, the small dimers will run much faster than the DNA fragments, so one can select the appropriate fragment sizes under a UV lamp.
6-Load all the samples on a 1.0% agarose gel, grouping them together on one side of the gel (you will need the vacant half later). Alternatively, two gels in the exact same buffer are employed. Buffer levels should be even with the top of the gel but should not flow over it. Fill in the unoccupied wells with buffer.
Keeping the buffer level below that of the wells reduces cross-contamination be tween the wells. Unoccupied wells must be filled with buffer to ensure the current runs evenly throughout the gel.
7-Run the gel for 60 minutes at 100 V.
8-Shut off the power, pipette off all of the buffer from each reservoir if you are going to re-use the gel. Stain the gel in Ethidium Bromide in 0.5 X TBE if you did not have EtBr in the gel itself. Take the gel to the darkroom for excision. Bring a razor blade.
9-View the gel on the UV transilluminator under the low power setting only. Photograph the gel for your records if you wish. Exposure to the UV light will cause pyrimidine dimerization, so view under low power as briefly as possible.
10-Using the lambda BstEII or other size standards as a guide, cut the region of the gel containing the fragments ranging in size from 0.7 to 2.0 kb. Remove this piece from the rest of the gel and set aside, keeping track of the orientation of the piece. Photograph the excised gel for your records. Return to your lab bench.
11-Estimate the size of the gel piece you excised and cut out a piece of similar size from the vacant half of the gel or from a second gel. Make this second excision 1-2 cm further from the wells than the first excision, and it should parallel the first excision so the first piece can be inserted in its original orientation.
12-Insert the first piece into this second hole. An exact fit is not required, as long as the excised piece sits easily in the hole, with the new front edge flush with the gel and filling any gaps with 0.5X TBE buffer.
13-Return the gel to the gel box if you are re-using the gel. Refill the buffer reser voirs until the buffer is just level with the top of the gel but does not flow across. Or lower the buffer if you are using a second gel. Fill any vacant wells with buffer. Fill any gaps surrounding the inserted gel piece with buffer. If the gel insert does not occupy the entire hole you made for it, be sure that at least the insert is flush with the side of the hole closest to the wells.
14-Reattach the electrical leads from the power supply in the reverse orientation as they were originally attached: i.e., the positive pole should now be closest to the wells.
15-Run the gel for the same amount of time, 60 min, and at the same voltage, 100 V, as you did originally. Re-stain the gel, if required. This procedure will pull the range of fragments back into a tight band. Subsequently, there is a smaller volume of agarose from which the DNA must be extracted.
16-Photograph the gel, if you want a record. Cut out the gel containing the con centrated bands and transfer to sterile microfuge tubes. If you have a large slice, you may wish to dice it before you transfer it to the tube, which will facilitate its solubilization in the subsequent steps.
17-DNA must be extracted from the gel. There are many procedures and commer cial products to isolate DNA from agarose: e.g., electrophoresis in dialysis tubing, Nucleotrap, Agarase, GeneClean, etc. Because DNA with short single-stranded extensions is very vulnerable to random nuclease digestion, we have opted for the fastest method: Qiaquick columns from Qiagen Corp.
18-Buy a Qiaquick kit from Qiagen and follow its directions. The final elution is in 50 µl of 0.01 M Tris, pH 7.5. Store Frozen (-70 °C).
Insert to Vector Ligation
You now have constructed insert and vector (both stored in aliquots at -70°C.) Congratulations! The remaining step is to ligate the two together. The adapted insert (50 µl) and vector (11 µl) are thawed on ice.
1-The following reagents, at ice temperature, are added to the DNA insert in the
following order.
The DNA insert and the vector are not in TE buffer. That much EDTA would very adversely affect the ligation reaction. EDTA will bind and remove Mg 2+ ion, and ligase requires Mg2+ for activity.
2-The reaction tube is placed at 16 °C for 6-8 hr. If all has gone well, you have constructed a library. To test the library, electroporate the DNA and titer the library for both opaque ("clear" or "white") and blue plaques. The number of plaques you need depends upon the average size of the inserts and the size of the original DNA.