DNA sequencing by partial ribosubstitution

J. Mol. Biob. (1978)

119, 83-99

DNA Sequencing by Partial Ribosubstitution WAYNE M. BARNEst Medical

Research Council Laboratoq of Molecular Biology Hills Road, Cambridge CB2 2QH, England

(Received 16 Jun,e 1977, and in revised form, 21 October 1977) A\ nets rapid m&hod for DNA sequence analysis leas been de\-iscd. In this ~uethod. base-specific cleavage is achieved at partially substit,utcd rihonucleotides \vhicll arc introduced by DNA polymerase extension in the presence of Mn2+. Access to a target sequence and label incorporation are achieved by extending a restriction fragment primer with DNA polymerase I. After a sllort initial incorporation wit11 [a-32P]deoxynucleotide triphosphates to label the 5’ region of tile target sequence, the triphosphates are removed and the reaction mixture is divided four ways for a second primed extension. The second extension is a and one of the four cold cl~asc in the presence of Mn2 + . all four tleoxyriilcleotities ri honucleotides under conditions that result in about 2”h ribonucleotides substitntion at eacll position. Aft’er cleavage at the restriction site and alkali cleavagr at, t tic positions of part.ial ribosubstitution, each reactiorl mixt8ure is analyscd b) vlrctrophorwis OIL a high-resolution denaturing acrylamide gel. As in the ottitrapid DKA srqucncing methods. the extent of DNA swiuence that, can be deterltlincd from a single experiment is limited only by the resolution of t,he analysing pcls. At, prcsrnt’ some 100 nnclrotidcs of scqwnc~~ ratI br det,crmincd from a sirigl(, priming rcwction.

1. Introduction DNA sequences has been made possible by restriction enzyme technology (for reviews see h’athans & Smith, 1974: Roberts, 1976). Priming of DIL’A polymerase synthesis by oligonucleotides or restrict ion enzyme-generated fragments of Dh’A has been used to direct label incorporation into specific target DNA regions for sequence analysis @anger et al., 1973; Maniatis et al.. 1974). The DNA sequence analysis was facilitated by efficient substitution of ribonucleotides by DNA polymerase extension in the presence of Mn 2+ . A rapid wav Do determine the nucleotide sequence of DXL4 labelled by primed synthesis, the “plus and minus” method, was devised by Sanger & Coulson (1975). B key observation made by Sanger a,nd his colleagues was that electrophoresis on acrylamide gels can resolve DNA molecules differing in length by a single nucleotide over the range of 20 to 140 nucleotides. if the molecules t’o bc analysed all have the same sequence and share the same 5’ end (Barrel1 et al.. 1976). Maxam & Gilbert (1977) USC similar high-resolution acrylamide gels in a DNA sequencing method that uses the principle of end-la,belling and basespecific part’ial chemical cleavage. Their technologr has the valuable capability of Easy

access to specific

identifying

reactiviby

the sites

for

binding

of proteins

of bound sequences towards

on DNA,

dimethyl

8::

k)y means

sulphatr

(Gilbert

of alteration

of the

et al., 1976). This

84

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paper describes a new procedure for rapid DNA sequence determination that utilizes some of the principles of each of these previous sequencing technologies. J. Dahlberg & J. Abelson (unpublished data) have suggested using the nicktranslation activity of DNA polymerase (Kelly et al., 1970) to introduce ribonucleotides into end-labelled double-stranded DNA, in an enzymatic variant of the Maxam & Gilbert (1977) method. This idea has not yet been exploited. The ribosubstitution approach described in this paper was successfully attempted in order to overcome an observed inability of the plus/minus method to obtain DNA sequence in regions of high twofold symmetry (protein-DNA interaction sites), while retaining the conveniences of the priming approach using DNA polymerase I extension (simple and easy labelling of target sequences, rapid procedures, no hazardous chemicals). In addition, this method was found to give more data about runs of nucleotides and to give rise to fewer artifact bands, in comparison with the plus/minus method.

2. Principle of the Method The principle of the method is illustrated in Figure 1. The method requires singlestranded DNA spanning the target sequence, and a restriction fragment primer must be annealed adjacent to the target sequence. DNA polymerase I is then used to

I, Primer

annealed

to template

5/L..-3’ target

2. Add slngk

3. Lobelhng

sequence

nbonucleotlds

extension:

wth

DNA

add o llmlted

p3lymerase

number

of [a-32P]dXTPs

4. Rlbosubstltutlon exterelon (4 separate reoctvznskchose dXTPs and one rXTP to partially ribosubstitute For instance, for the As

wth

5’--rC....*-rA -

rC...n-

rA-

-

rC.....-rA

5 Cleave at the ribonucleotides and electrvphorese mMx’e on denoturnq ac.rylamKie gel A

G

C

T t C T 8

D-l4

FIG.

1. Summary

i

of the method.

each reoCtlCn

4

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SEQUENCING

METHOD

X.5

extend the primer under carefully controlled conditions. After addition of a single ribonucleotide (N. L. Brown, unpublished data), a limited number of radio-labelled deoxynucleotides are introduced in what is called the labelling extension. The length of the radioactive extension is controlled by limiting the time and temperature of the reaction or, more easily, by limiting the nucleoside triphosphates supplied. At least, some molecules should only be extended for a short distance, since no sequence can he deduced before the end of the shortest of these labelled molecules. After removal of the triphosphates by gel filtration, the DNA4 solution is apportioned into four partial ribosubstitution reactions. Each of these reactions is an unlabelled extension in the presence of Mn2 + , all four deoxynucleotide triphosphates and one ribonucleotide triphosphate. The ratio of ribonucleotide concentration to tha,t of its corresponding deoxynucleotide is carefully adjusted to result in about 2”/,, ribonucleotide substitution at each position for that, base in the DNA sequence. Suitable ratios can be found for all four bases. Chemical or enzymatic cleavage at thtk ribonucleotides then results-for the rA/dAt reaction, for instance-in a collection of labelled molecules whose length is a measure of the position of A residues in the sequence. High-resolution electrophoresis on an acrylamide slab gel is us?d to analyse clach of the four reaction mixtures in different columns on t’he gel. Autoradiography of the gel results in a band for each base in the appropriate column. and the sequence (aan ht. read off directly.

3. Materials and Methods (a) Chemicals and label Ultra-pure urea \vas purchased from Sohwarz-Mann. All other chemicals were reagent, grade purchased from BDH or Sigma. Nucleoside triphosphates were from Boehringer Mannheim or PL Biochemicals. [a-32P]deoxynucleosidc triphosphates at, a spec. act. of 100 to 150 mCi/pmol were from New England Nuclear.

(b) Enzymes Restriction enzyme HaelII from Haemophilus aegyplius was a gift from Nigel L. Brown. or was bought from Miles Laboratories. Escherchia co& DNA polymerase I and it,s large fragmcmt (Brutlag et al., 1969; Setlow et al., 1972; Klenow et al.. 1971) were bought, from Boehringer. (c) DNA

Phage 4X 174 RF DNA was prepared with C. A. Hutchison III or was a gift from 3. Drouin. Bacteria (E. coli C) were infected with lysis defective phage (am3) in the presence of chloramphenicol (Hutchison & Sinsheimer, 1966), and the supercoiled RF (replicative form) DNA was isolated by the cleared lysate method (Katz et al., 1973) just aa for plasmid DNA (see below). +X plus strand (virion) DNA was a gift from C. A. Hutchison. DNA plasmid pWB91 carries the His OGD region of Salmonella typhimurium and the co1 El replicon. It was constructed from Ah80dhis (Voll, 1972) DNA and mini El (Herschfield et al., 1974) DNA by recombination ilz vitro, as described by Herschfield et al. (1974). The plasmid contains a single site for restriction enzyme EcoRI. Details of this plasmid’s structure and sequence will be described elsewhere when known. pWB91 DNA was prepared from saturated cultures of bacteria by the cleared lysate method (Katz et al., 1973), including 2 equilibrium bandings in CsCl/ethidium bromide gradients. Separated strands of pWB91 DNA were prepared from EcoRI-digested linear molecules by the poly(U,G) method of Szybalski et al. (1971). The fractions of separated strands from the CsCl buoyant density gradients were self-annealed at 68°C for 30 min and rebanded in t Abbreviations used: rX, ribonucleotide; dX, deoxynucleotide; A, adenosine mononucleotide U triphosphate, as appropriate, and similarly for G, guanosine; C. cyt,osine; T, thymidinr: or. uracil.

86

W.

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CsCl to remove contamination of opposite strand. The twice-banded DNA strands were dialysed, precipitated with ethanol and resuspended in water. No effort was made to remove poly(U,G). (d) Restriction fragments Preparative restriction enzyme digests were fractionated on 5% to 20% acrylamide gradient gels (Jeppesen, 1974), 40 cm in length. The bands were stained with ethidium bromide (0.5 pg/ml) and visualised under shortwave 11.v. light (Sharp et al., 1973). The DNA was eluted electrophoretically by the following procedure. Each gel slice and 2 to 3 ml of elution buffer (0.1% sodium dodecyl sulphate, 10 mM-Tris-acetate (pH 8.3), 4 mMsodium acetate, 0.4 rnM-EDTA) were put into a dialysis bag tied at both ends with no entrapped air. Several such dialysis bags were then placed in a parallel configuration in a small (15 cm x 10 cm x 10 cm) plastic container and covered with 2 cm of elution buffer. Platinum-clad electrodes were immersed in the buffer at the sides of the tray parallel to the dialysis bags and a current of 40 mA was applied across the ba,gs and gel slices for 12 h, after which time the DNA was out of the acrylamide but still within the dialysis bags. The current was reversed for 15 min. The bag contents were then filtered through glass wool and rendered 0.3 M in ammonium acetate (pH 5.2) and 75% in ethanol for precipitation. If the volume was much larger than 2 ml before adding acetate and was concentrated by ethanol, the solution was made only 0.1 M in ammonium extracting twice with an equal volume of 2-butanol (Baum & Gesteland, unpublished data) (discard upper phase; ethidium bromide is removed at t,his optional step). One extraction with CHCl, removes 2-butanol from the aqueous phase before precipitation with ethanol. The fragments were pelleted by centrifugation at 8000 g for 10 min in siliconised glass tubes. The DNA fragments were resuspended in water and stored frozen. The recovery was 50 to SO%, as judged from ethidium staining intensity of small samples of pure fragments electrophoresed on acrylamide or agarose gels. Some purified $X fragments used were a gift from N. L. Brown. (e) Sequencing procedure The following basic procedure was followed except as noted in the Figure legends. The example described is a priming with HaeIJI restriction fragment 3 on $X174 plus-strand template. The appropriate initial ribonucleotide here is rC (two rCs are incorporated since the HaeIII cleavage site is G-G 4 C-C). (i) Addition of an initial ribonucleotide (e.g. rC) (modijed from N. L. Brown, unpublished data). 1 pmol of $X174 plus strand and 1 pmol of restriction fragment primer in 14 ~1 water were mixed with 2 ~1 of concentrated annealling buffer (0.1 M-Tris.HCl (pH 7.4), 10 mMdithiothreitol, 0.5 M-NaCl) sealed in a capillary, heated 3 min at 100°C to denature the DNA, and incubated 30 min at 67°C to reanneal. The annealed DNA solution was then mixed with 2 ~1 6.7 mm-MnCl, (0.67 mM final), O-5 ~1 mM-rCTP (25 pM final) and 1 ~1 (1 unit) DNA polymerase I, and incubated for 10 min at 20°C. (ii) Labelling extension The label was then introduced using one of the following 3 procedures to extend the DNA for a limited distance. Each of the 3 possibilities includes 25 PM-dCTP to chase the (pH 7.9), 10 mM-MgCl,, initial rC in t,he presence of Mg 2+. TMD buffer is 10 rnwI-Tris.HCl 1 mM-dithiothreitol. 2 x TMD is twice concentrated TMD. (1) Three to twenty labelled nucleotides. This method was used for the examples shown in Figs 2 to 4. In this method of labelling only 3 or fewer dXTPs are supplied and label is incorporated over the distance to the first position requiring the missing nucleotide(s). Even without knowledge of the initial sequence adjacent to the primer, an empirical test can determine which combination of labelled dXTPs will put the most label into a limited 5’ region of the target sequence. In the example sequence shown in Figs 2 to 4 there are no is appropriate. Ts for 18 nucleotides after the primer, so “minus T” labelling To the completed initial rC reaction is added an equal volume (20 ~1) of 2 x TMD buffer containing 0.5 X lo-lo mol [a-32P]dATP and [a-32P]dGTP and 1 ~1 of 1 mivr-dCTP (25 pM

A RAPID

DNA

SEQUENCING

METHOT)

Xi

final). After 20 ruin at 37”C, t’he reaction is stopped wit,11 3 pl of 0.5 M-EDTA and thts triphosphates are removed by gel filtration on a 1 ml GlOO Sephadex column in a 1 ml thin-walled disposable pipette (GlOO buffer is 2.5 mu-Tris-HCl (pH 7.4), 1 mM-NaCl). The DNA peak, detected with a hand radiation meter, is evaporated to dryness under vacuum. (2) Limiting substrate extension. The rationale of this method of labelling is that extrxndinp molecules terminate as limiting substrai,e dXTPs arc‘ used up. In this case t hf. limitilrg dXTPs arc also the (a-32P)-labeIled ones. The completed initial rC reaction is divided into 2 portions. To eacll portion is added WI equal volume of 2xTMD buffer containing 50 PM-dCTP plus either 4 or 10 pmol (a-32P)-labelled dTTP, dGTP and dATP. After 10 min incubation at 20°C the 2 rractions WV pooled with 3 ~1 0.5 M-EDTA and gel-filtered on Cl00 as for (1) above. (3) Limiting label extension with time and temperature @anger & Co&on, 1975). To the in&t1 rC reaction is added an equal volume (20 ~1) of 2 x TMD buffer containing 1 ~1 of I InM-tlCTP and 1. 2 or 3 (cc-32P)-labelled dXTPs (1 PM final) and 2, 1 or 0 complementing Irnlabrlled dXTPs (10 PM final). Incubation at 4°C is carried out for 1 min for half t’hr> rc,act,ion mixture and 3 min for the other half. Each reaction sample is stopped by adding it to 3 ~1 0.5 M-EDTA. GIOO gel filtration of the combined samples is applied according t 0 ( 1 ) RhO\X’.

I i Ii ) Second extension (partial

ribosubstitution)

Partial ri hosubstitution was accomplished in the second extension. Stock ribonucleotidei tlc,oxyrlucleotide solutions containing 4 dXTPs and one rXTP were made at 10 times the final concentrations shown in Table I and stored frozen in water. Ten-times concentrated (pH 7.4), 0.67 mYIribosubstitution buffer (Salser et al., 1972; final 1 x = 67 mM-Tris.HCl MnCl,. 10 mM-p-mercaptoethanol) was made up fresh immediately before use. The DNA from the labelling extension was resuspended in 4 ~1 of 10 x ribosubstitution t,llffer plus 28 ~1 wat)er and divided into 4 portions of 8 ~1. To rach portion in a capillary TABLE

1

Ribonucleotideldeoxynucleotide .\. “Low”

I~ih~~nucl~otide/deoxynucleotide I+‘inal r/d ratio ELM-ribopwdeoxy-

rA/dA

B. “High”

ribonuclrot,ide/deoxynucleotide Final r/d ratio p&r-ribsPM-thxy-

ratio rG/dG

IC/dC

10 100 10

3 30 10

150 100 0.67

The other three dXTPs

concentrations

260 100 0.4

are at 10 PL”

ratio

rA/dA

rG/dG

rC/dC

500 100 0.2

100 100 1

10 100 IO

Thr ot,hrr three dXTPs

rU/dl

rU/dT 1000 200 0.2

are at 10 p’~.

These are t,he concentrations of ribo- and deoxynucleoside triphosphates employed in the partial ribosubstitution reaction. (A) The low ribonucleotide/deoxynucleotide ratios give rise to relatively infrequent ribosubstitution and are appropriate when the labelling extension is short. (B) The high ribonucleotide/deoxynucleotide ratios are appropriate when a spread in molecular lengths has already been achieved at the Iabelling extension.

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M. BARNES

was added 1 ~1 of the appropriate 10 times concentrated ribonucleotide/deoxynucleotide solution (see Table 1) and 1 ~1 (1 unit) DNA polymerass I. After incubation at 37°C for 30 min, 10 ~1 of all 4 dXTPs at 50 pM in 2 x TMD were added and the reactions were incubated for a further 30 min at 37°C. The utility of this dXTP chase step is illustrated in Pig. 3 (see Results). The ribosubstitution reactions were stopped by adding 2 ~1 of 0.5 M-EDTA, and then evaporated to dryness under vacuum. (iv) Enzyme cleavage If the initial ribonucleotide step was not used to provide recleavage at the restriction site, restriction enzyme cleavage could be carried out simultaneously with the above dXTP chase step. Some restriction enzymes are partly inhibited by the Mn2+ and require a longer than normal incubation.

(v) Ribonucleotide cleavage Chemical cleavage at the ribonucleotides was carried out by hydrolysis in piperidine (Book, 1967). Each dried sample was resuspended in 20 ~1 of lOe& freshly diluted piperidine, sealed in a capillary and incubated for 3 to 16 h at 67°C. After this time the samples were removed from the capillaries and evaporated to dryness under vacuum 3 times to remove piperidine (1 drop of water was added between evaporations). (f) High-resolution

gels

The high-resolution acrylamide gels are only slightly modified from Sanger & Coulson (1975), Air et al. (1976) and Msxam & Gilbert (1977). The gel composition is 20% acrylamide, l/60 bisacrylamide (0.33%) in electrophoresis buffer (60 mM-Tris-borate (pH 8.3), 1 mivr-EDTA). Gel dimensions are 20 cm x 40 cm x 1.6 mm. Samples are loaded in 10 to 15 ~1 of 90% formamide, 10 mm-EDTA, 0.020/b xylene cyan01 and 0.03% bromphenol blue. They are heated for 30 s at 90°C and cooled immediately before loading. Samples may be split and portions electrophoresed for various times, until the xylene cyan01 has moved 25 cm (-7 h), 50 cm (-14 h) or 75 cm (-21 h). Electrophoresis is carried out at about 1000 V and 20 to 25 mA, so that the gel warms up to 40 to 60°C as a result of electrical heating. This heating serves to denature most of the secondary structure in the DNA strands being analysed (Sanger $ Coulson, 1975).

(g) Autoradiography The gels are soaked in 1076 acetic acid for 15 min to fix the DNA (Air et aZ., 1976). They are then rinsed briefly in water and blotted to remove excess liquid, and the wet gels are wrapped in Saran wrap for autoradiography with X-ray film at room temperature.

4. Results The sequence adjacent to the $X174 Hue111 fragment 3 was chosen to test the method. Priming was carried out on viral plus-strand template, so the sequence deduced corresponds to that of the minus strand. This sequence was already known from the work of N. L. Brown & M. Smith (unpublished data) and Sanger et al. (1977). (a) Pirst extension labelling of the example sequence which makes it useful as a test sequence is that no Ts in the first 18 nucleotides adjacent to the primer. Thus the initial extension can be effectively limited by leaving dTTP out of the reaction. The result of such a labelling extension can be seen in the channels labelled 0 in Figure 2, which were not subjected to the ribosubstitution extension, but were otherwise treated identically and cleaved at the initially inserted rCs. It can be seen that not all of A feature

it contains

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89

the molecules stop before position 19. Some 50% continue on to stop in front of the second T at position 29. I do not know why the first T is partially read through; but two possibilities suggest themselves. This read-through may be caused by contamination of the reagents with dTTP, or alternatively, the polymerase may misincorporatc some dCTP at position 19. A useful amount of label (l/2 &i) was readily and specifically incorporated at this step using 2/3 pmol of DNA and 10 PCi of [a-32P]dATP and tiGTP. Since no sequence can be deduced using this technique from the region shorter t,han the shortest labelled molecules from the labelling extension, in this example t,he first 18 nucleotides of sequence would not be determined.

(k)) Ribonucleotide~deoxynucleotide ratio In previous studies of ribosubstitution by E. coli DNA polymerase I (Berg et al., 1963; Van de Sande et al., 1972), the differing abilities of each base to be ribosubstit’uted have been observed to be C N G > A >> T(U), although previous studies did not find that rU could be incorporated to any useful extent. Part,ial ribosubstitut,ion in the presence of all four deoxynucleotide triphosphates is a very sensitive indicator however, and a preliminary experiment by J. of ribonucleotide incorporation, Dahlberg (unpublished data) showed that each of the four bases can be partially ri bosubst’it’uted a6 some ratio of rihonucleotide triphosphate to deoxynucleotidc triphosphate. The effect, of varying the ratio of ribotriphoaphate to deoxytriphosphate is demonstrated for each base in the experiment shown in Figure 2. A constant concent,ration (0.1 mM) of rNTP was used. It can be seen that for each base there is a high ratio at which the ribosubstitution is too efficient, and the sequence more than 60 nucleotides from t,he primer cannot be determined, while at a low ratio the ribosubstitution is not efficient enough. The optimum ratios for ribosubstituting DNA which has been labelled by a very short labelling extension can be seen to be about ribo/deoxy = I50 for A. 10 for G, 3 for C and 250 for T(U). These ratios inversely reflect the differing a,bility of each base to be ribosubstituted by DNA polymerase 1. If an alternate procedure is used for the initial label incorporation such that a wide range of extensions is achieved at the labelling step (see Materials and Methods), tfhe high ribo/deoxJr ratios arc more appropriate at the ribosubstitmion extension.

(c) Initial

ribonucleotide

The advantage of using an initial fully substit,uted ribonucleotide in the restriction site (N. L. Brown, unpublished data) can be seen by comparing Figures 3 and 4. In the experiment shown in Figure 3, final cleavage at the primer restriction site was carried out enzymatically with enzyme HaeITT. The cut-off at 72 bases in Figure 3 is at the next recognition site for enzyme HaeIII. The experiments in Figures 2 and 4 (and 5) utilized the initial ribonucleotide to supply the final cleavage at the restriction site. Additional advantages to the initial ribonucleotide include a saving in time and expense of digestion by restriction enzyme. Also, it has been observed that restriction enzyme Ah1 is inhibited by single-stranded DNA and it is t’herefore not, possible to use AZuI to digest a short primed extension on a single-strended template. The initial ribonucleotide incorporation is therefore the necessary method of recleavage at AZuI sites (N. L. Brown, unpublished data). At some restriction sites there is a potential problem with the initial ribonucleotide incorporation which is well illustrated by the channels labelled 0 in Figures 2 and 4.

90

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29

-19

FIN. 2. Effect of varying the ribonucleotide/deoxynucleotide ratios. This experiment, which uses 4X plus strand and Hoe111 fragment 3 as primer, followed the protocol described in Materials and Methods with the following particulars: two ribooytosine residues were initially incorporated on the end of the reatrict,ion fragment primer, followed by limited label incorporation 8s described in Msterials and Methods (“minus T” lebelling). At the partial ribosubstitution incorporation, the

2-i RAPID

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METHO

Since the restriction site being utilized is an Hue111 site (recognition sequence G-G-C-C). two ribonucleotides must initially be incorporated. To the extent that only one of the rCs is incorporated, all of the bands on the gel will be double. In the experiments shown in Figures 2 and 4, some 3 to 10% of the second cytosine residue were not ribosubstituted, and this has caused a 3 to 10% (visually estimated) shadox% above each of the bands on the gel. This does not interfere seriously with interpretation of the sequence except for some of the A residues (see below). (d) “Chasing

out” a&e&t

bands

The importance of chasing the ribosubstitution extension reaction by final incubation with excess deoxynucleotide triphosphates is demonstrated in Figure 3. After the ribosubstitution extension, 10 PM of each dXTP was included during the Hue111 recleavage in the reactions analysed on the right half of the Figure, but no such chase was carried out in the reactions in the left. It can be seen that various artefact bands are present in the left half of the gel, such as the one in the T(rU) channel at position 23. These bands are not the result of ribocleavage at their 3’ end. Rather, they result, from premature termination of DNA polymerase extension. Whatever their cause. these bands have been chased into full-length (72 bases) DNA in the right half of t’hc
T-g-G-G

“missing

Gs”

position

2

T-&C,

3

Aa,

“weak

following

4

cc,

“weak

initial

G)

As”

Cs”

relevant ribonucleoside triphosphate was present at nucleotide ratios shown were achieved by varying the nucleoside triphosphate. The outside channels, labelled subjected to t,he ribosubstitution extension or its chase, the initially incorporated ribonucleotides. The analysing on the right measure the nucleotides from the HneIII SC‘, xS’lene rvanol.

52

positions

33, 47

positions

22-25, 44, 70

positions

30, 73

100 P”I, and the ribonucleotide/deoxyconcentration of the homologous deoxy“O”, contain material which was not but this material was cleaved normally at gel was 14o/o acrylamide. The numbers restriction site. RPB. bromphenol blue;

W. M. BARNES

92

rA

rG

rC

rU

r

rA

C hayd rG rC

rl J

\

FIG. 3. This gel illustrates several variations on the standard procedure. (1) The left half of the gel illustrates the effect of omitting the final dXTP chase. (2) The r/d ratios used were A = 260, G = 10, C = 3, T(U) = 600. (3) The acrylamide gel is 14% acrylamide in TBE buffer (89 mMTris . borate, pH 8.3, 2.6 m&t-EDTA). The sequence shown is the same as that in Figs 2 and 4. XC marks the position of the xylene cyan01 dye. The numbers on the right measure the nucleotides from the Hoe111 restriction site. The top of the gel was cut off before autoradiography.

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The weak bands are worse (weaker) for the sequences at the top of the list. The relative strength of the weak bands is unchanged when pancreatic ribonuclease (for c,s and Us) and T, ribonuclease (for Gs) and T, ribonuclease (for As) are used t’o cleave the ribonucleotides (results not shown). It can be seen in Figure 2 that’ t,he weak G in T-g-C-G shows up a little better when the ribo/deoxy ratio is 100 instea,d of 10 (po&ions 33 and 67). The example sequence is unusually rich in t’he troublesomr t,etramer T-g-C-G. I have also observed (data not shown) that the weak G in T-g-C-C: is more efficiently ribosubstituted when the buffer contains less Tris.HCl (20 maI). However, enzymatic processing of the first extension is incompl-te in this buffer. wit)h t,he result that unextended molecules (“0 bands”) appear across all channels of t,hc gel at positions 19 and 29. (f) Sequencing of cloned DNA $X174 is a convenient template for the experiments shown in Figures 1 to 3 because of its small size and easily available single-stranded DNA (the virion DNA). Many objects of DNA sequencing are likely to be cloned on a plasmid. Figure 5 illustrates the applicability of the partial ribosubstitution technique to DNA cloned on a plasmid. The sequence shown is from the promoter and genetic control region of t,he histidine operon of Salmonella typhimurium. This sequencing gel is presented as an example. Details of the histidine plasmid and its sequence will be published tllsewhere when they are complete.

5. Discussion (a) Data interpretation I have described a simple and rapid technique for DNA sequence analysis. The data are produced in an easily interpretable form : there is a band in an autoradiograph t)o identify the base at each position in the sequence. As demonstrated in Figure 4, at least 80 bases of sequence can be read from a single experiment, corresponding to positions 20 to 100 adjacent to a primer restriction fragment. The data in Figure 4 for positions 100 to 150 can (if the gel is electrophoresed for a longer time) complement and confirm other incomplete DNA sequence data, such as that determined from the amino acid sequence of a coded protein. The only problem with the data generated by this method is a result of a previous13 unsuspected property of the ribosubstitution reaction catalysed by DNA polymerase 1: the frequency of partial ribosubstitution has a sequence-specific variability, with some positions in the sequence being only weakly ribosubstituted relative to t’he others. This causes weak or even missing bands in the final gel. The effect is not’ random, however, and appears to be sequence specific at the tetranucleotide level for the very weak bands (T-g(C, G) and T-g-G-G) and at the dinucleotide level for thr mildly weak bands (CC, and Aa,). Since all 256 possible tetranucleotides have not yet been test-sequenced with the method, and since this problem could not have been anticipated, other problem t’ctramers may yet turn up. Actually, once the regularity of the mildly weak bands has been recognized, their very weakness provides redundant sequence information during interpretation of the sequence. For example, a weak A band immediately above a strong A band indicates that there is definitely no other base between the weak A and the first A.

0

A

T

G

C

860

50

-40

32

.22 FIG. 4. The standard sequencing procedure applied to priming with +X174 Hue111 fragment 3 on the viral strand template. Two initial ribocytidine residues were incorporated followed by labelling in the absence of T as described in Materials and Methods. The material run in the channel labelled “0” was not subjected to the ribosubstitution extension, but was treated with piperidine as normal. The numbers on the right measure the nucleotides from the Hue111 restriction site. The top of the gel was cut off before autoradiography. Sequence interpretation: the two bands in the 0 channel represent the starting material for the ribosubstitution step. Some of this material has not been processed in t,he ribosubstitution extension and remains across the other channels as artefact bands in the other channels, particularly in the C channel. The sequence oan be read without difficulty, ambiguity or error until position 106 with the following possible exceptions: thr G hands at positions 33, 47, 52 and 99 arc not invisible but,

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95

METHOD

The mebhod as here demonstrated does not generate sequence data closer than 10 to 15 nucleotides t’o the primer restriction site. It is likely that the incorporation of one to three [a-32P]dXTPs would provide adequate and specific label to allow partial rillosuhst,itut’ion (and thus sequence data) much closer to the primer site, but’ t’his has not ;c+ heen demonstrated. (b) General strategy The technique requires single-stranded DNA spanning the target region. If t,htt t.arget, DNA is carried on bacteriophage lambda or on co1 El plasmid as a vector. the DNA strands can usually be separated by the poly(U,G) binding technique of Szybalxki PI al. (1971). I have found, in experiments with a mini El-histidine plasmid DN1\ (Fig. 5 and W. M. Barnes, unpublished data), that the poly(U,G) need not be degraded before using the strands in a sequencing experiment. Separation of DKA strands b,v gel elrctrophoresis is not suitable as a preparative method, since high concentrat’ions of DNA do not remain denatured as they enter the gel (B. GriRin. unpublished data). This sequencing technique also requires the identification and purification of rclstriction fragments adjacent to the target region. Of the available techniques for rcbstriction sit’e mapping, the pulse-chase priming procedure of Jeppesen et cd. (1976) and Summers (1975) is most compatible with t,his sequencing technique, since it’ has the same physical requirements in terms of DNA substrate. chemical substrate (I a-32P]dXTPs) and enzyme tools (DNA polymerase I). Similarly, the mapping tcbchnique of Smit#h & Birnstiel (1976) is directly compatible with the sequencing tt~chnology of Maxam & Gilbert (1977). The strategy of how to carry out the labelling extension depends on hou- close to the primer t,he desired sequence is located. For nucleotides 15 to 40, only 15 or fewer In hc~llod nucleotides should be inserted at the first extension using a variation of the “minus I”’ labelling procedure described in Materials and Methods. Limitation of iuit’ial label by limiting substrate or time and temperature (see Materials and Methods) tt>nds t,o miss out the first 30 nucleotides, but provides ample label for sequencing out t,o the limit’ of gel resolution. In this case high ribonucleotide/deoxynucleotide ratios would be appropriate during the partial ribosubstitution reaction, since the spread in molecular lengths is achieved at the labelling extension. (c) Advantages

over other rapid methods

Although application of the plus and minus method (Sanger & Coulson, 1975) hss recently resulted in the complete sequence of +X174 (5400 nucleotides, Sanger ef al., 1977). data from this method needed to be backed up by data from amino acid

the>- are particularly weak; t,he C band at posit,ion 74 is particularly strong and is overexposed on this autoradiograph and obscures C73. All of the other bands up to position 112 are resolved on the original autoradiograph although some resolution has been lost in t,his reproduction. The *cy”errc~e 1s: .T GAAAAAGCGT 20 TGTAACCAT~~ 60 C’GT.. 110 Hyphens

CCTGCGTGTA 30 AGGCCACGTA 70

have been omitted

to *ax-e space.

GCGAACTGCG 40 TTTTGCAilGC 80

ATGGGC’AT.-\(! 50 TATTTAACTG 90

GCGGCGATTG 100

96

W.

0

M.

A

BARNES

T

G

C

860

50

-40

-32

-22 FIG. fragm

of1

Dartial ribosubstitution to a cloned DNA sequent )lication a mini-El-histidine used to prin ne on the H strand of pWB91,

An H id

striction Gng part

.4 RAPID

DNA

SEQUENCING

97

METHOD

sequence (Air et al., 1976), depurination data (N. L. Brown & M. Smith, unpublished data), partial exonuclease digestion and RNA transcription, due to several problems. These problems, which have been largely overcome by the partial ribosubstitution t,echnique, are listed below in decreasing order of importance. (1) The plus a.nd minus method requires a uniform spread of molecular lengths resulting from the labelling extension. Protein recognition sites in interesting DNA control sequences often have partial twofold symmetry (Gilbert BE Maxam, 1973 ; Dickson et al., 1975: Maniatis et al., 1975). Such symmetrical sequences give rise in the single-stranded DNA template to secondary structure which blocks smooth extension by DNA polymerase at the labelling extension. This results in an irregular labelling extension product which the plus and minus method cannot adequately process for a sequence. This problem occurred only once in the 5400 nucleotides of 4x174 (Fiddes, 1976), but it is much more frequent in DNA containing genetic control sites. 1 have encountered this problem at two places within 200 base-pairs in the histidine operon genetic control region, and this was the reason for attempting the parbial ribosubstitution approach described here. The partial ribosubstitution procedure was found to thrive on a,n uneven labelling extension. thus completel? c~liminnting this problem. (2) Artefact bands, which occur with significant frequency in t#he plus and minus method, are rarely seen in the partial ribosubstitution method, and when they art’ they can be easily recognized as left-over molecules from the labelling extension. 1 have also noted that the band intensities are much more uniform in the part,ial rihosubstitution procedure, allowing improved signal/noise discrimination. (3) Runs of nucleotides. such as A-A-A-A-A, appear as gaps in the plus and minus method. In the partial ribosubstitution method every base in the run gives rise t,o a hand (except for T-g-G-G). The partial ribosubstitution procedure is similar in principle to the chemical basespecific cleavage method of DNA sequencing (Maxam & Gilbert, 1977), and it offers: in principle, no advantages over the chemical method in terms of the data and information generated. The partial ribosubstitution mebhod does have several pract,ical procedural advantages, however, once single-stranded DNA template and restrict,ion fragment primer are at hand. (1) This method is more rapid and simple than the chemical base-specific cleavage procedures. A single experiment takes one day instead of Owe to t,hree days to pcrform. Much of the comparative saving in time is achieved at the labelling step since only one sequence is labelled by the DNA polymerase extension. The chemical method requires a separation of two DNA fragments or strands after the labelling step of each sequencing experiment. (2) Ko hazardous

chemicals,

such as dimethyl

sulphate

or hydrazine,

are employed.

of the 8. typhimurium. histidine operon. The strands of this plasmid were separated by the, poly(U,G) technique of Szybalski et al. (1971). The separated strands were self-annealed and rebanded in caesium chloride, but no other effort was made to remove the poly(U,G). The labbelling extension was carried out in the absence of A. The position numbers shown are only estimates, due to lack of knowledge of the sequence immediately adjacent to the Hha restriction site ut,ilized. The sequence reads (hyphens omitted) : AAAAGTGGTT 20

TAGGTTAAAA 30

GGTATCAAAT

GAATAAGCAT

TCGATCGGAA

TTTTT..

40

50

ti0

70

98

W.

M. BARNES

(d) Future possibilities (1) It should be possible to eliminate the need to identify and purify the fragment adjacent to the target sequences. A multiple priming with many restriction fragments annealed to a single strand at the same time should give rise to many 5’-labelled, partially-ribosubstituted restriction fragments at once. In preliminary experiments I have found that such fragments can then be purified by gel electrophoresis, treated with alkali, and analysed directly on high-resolution sequencing gels to generate sequence data. (2) Since it is possible to cleave the ribosubstituted nucleotides in the doublestranded DNA with pancreatic ribonuclease (for U and C), bound genetic control proteins such as RNA polymerase or repressor should protect their recognition sites from ribonuclease attack, thus identitying the recognition sequences. This work was carried out in the laboratory of Fred Sanger, whom I thank for his encouragement and interest. The author was supported by post-doctoral fellowships from the American Cancer Society (grant no. PF-1020) and the National Cancer Institute, (grant no. 1 F32 CA0586501.) REFERENCES Air, G. M., Sanger, F. & Coulson, A. R. (1976). J. Mol. Biol. 108, 519-533. Barrell, G. G., Air, G. M. & Hutchison, C. A. (1976). Nature (London), 264, 34-41. Berg, P., Fancher, H. & Chamberlin, M. (1963). In Symposium 0% Informational Macromolecules, p. 467, Academic Press, New York. Bock, R. M. (1967). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 12A, pp. 224-228, Academic Press, New York. Brutlag, D., Atkinson, M. R., Setlow, I?. & Kornberg, A. (1969). Biochem. Biophya. Res. Commun. 37, 982-989. Dickson, R. C., Abelson, J. N., Barnes, W. M. & Reznikoff, W. S. (1975). Science, 187, 27-35. Fiddes, J. C. (1976). J. Mol. Biol. 107, l-24. Gilbert, W. & Maxam, A. (1973). Proc. Nut. Acad. Sci., U.S.A. 70, 3581-3584. Gilbert, W., Maxam, A. & Mirzabekov, A. (1976). In Control of Ribosome Synthesis (Kjeldgaard, N. 0. & Maalae, O., eds), pp. 139-148, Munksgaard, Copenhagen. Herschfield, V., Boyer, H. W., Yanofsky, C., Lovett, M. & Helinski, D. R. (1974). Proc. Nat. Acad. SC;., U.S.A. 71, 3455-3459. Hutchison, C. A. & Sinsheimer, R. H. (1966). J. Mol. Biol. 18, 429-435. Jeppesen, P. G. N. (1974). Anal. Biochem. 58, 195-207. Jeppesen, P. G. N., Sanders, L. & Slocombe, P. M. (1976). Nucl. Acids Res. 3, 1323-1339. Katz, L., Kingsbury, D. T. & Helinski, D. R. (1973). J. Bucteriol. 114, 577-591. Kelly, R. B., Cozzarelli, N. R., Deutscher, M. P., Lehman, I. R. & Kornberg, A. (1970). J. Biol. Chem. 245, 39-45. Klenow, H., Overgaard-Hansen, K. & Patkar, S. A. (1971). Eur. J. Biochem. 22, 371-381. Maniatis, T., Ptashne, M., Barrell, B. G. & Donelson, J. E. (1974). Nature (London), 250, 394-397. Maniatis, T., Jeffrey, A. & Kleid, P. G. (1975). Proc. Nut. Acad. Sci., U.S.A. 72, 1184-1188. Maxa.m, A. N. & Gilbert, W. (1977). Proc. Nut. Acud. Sci., U.S.A. 74, 560-564. Nathans, D. & Smith, H. 0. (1974). Annu. Rev. Biochem. 44, 273-293. Roberts, R. J. (1976). CRC Crit. Rev. Biochem. 4, 123-164. Salser, W., Fry, K., Brunk, C. & Poon, R. ( 1972). Proc. Nut. Acad. Sci., U.S.A. 69,238-242. Sanger, F. & Coulson, A. R. (1975). J. Mol. Biol. 94, 441-448. Sanger, F., Donelson, J. E., Coulson, A. R., K&sel, H. & Fischer, D. (1973). Proc. Nat. Acud. Sci., U.S.A. 70, 1209-1213. Sanger, F., Air, G. C., Barrell, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., Slocombe, P. M. & Smith, M. (1977). Nature (London), 265, 687-695.

A RAPID

DNA

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Setlow, P., Brutlag, D. & Kornberg, A. (1972). J. Biol. Chem. 247, 224-231. Sharp, P. A., Sugden, B. & Sambrook, J. (1973). Biochemistry, 12, 30553063. Smith, H. 0. & Birnstiel, M. L. (1976). ZVucZ. Acids Res. 3, 2387-2399. Summers, J. (1975). J. Viral. 15, 946-953. Szyhalski, W., Kubinski, H. & Summers, W. C. (1971). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 21D, pp. 383-413, Academic Press, New York. Vttn de Sande. J. H., Loewen, P. C. & Khorana, H. G. (1972).J. Riol. Chem. 247, 6140-6148. \ToII. M. .J. (1972). J. Bacterial. 109, 741-750.