[17] A sytematic approach to chemical DNA sequencing by subcloning in pGV451 and derived vectors

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231

those with the 3-kb band. Similarly, the frequency of finding the 2- and 3kb bands is greater than finding the 1-kb band. The average length of the sequence that could be read from the 80-cm gel with one loading of 32p-labeled DNA was 150-250 bp; up to 450 bp could be read with two loadings. Thus, plasmids with deletion approximately 200-400 bp apart in the pAA-PZ derivative were chosen for DNA sequence analysis. If 35S-labeled DNA is used, it is likely that a longer sequence could be obtained. We found that certain DNA sequences when cloned into pAA-PZ vectors tend to increase the rate of in oivo deletion. Thus, in some cases, it may not be possible to get the partially deleted clones with desired end points. We also found that transposon-promoted deletions exhibited a considerable amount of sequence specificity. In some cases, a higher percentage of the deletion end points tended to terminate around G : Crich regions. In other cases, deletion end points cannot be obtained within a region of 900 bp. The problem that deletion end points may miss a certain region of the foreign DNA to be sequenced could be solved in one or two ways: either by analyzing more clones or by running an identical gel for a longer time. The latter method would be equivalent to loading a third sample of DNA on an 80-cm-long gel. In this case, up to 700 bp could probably be read by using 35S-labeled DNA. Acknowledgments The work was supported in part by Research Grant GM29179-06 from the National Institutes of Health, United States Public Health Service, and by Research Grant RF 84066, Allocation No. 3, from the Rockefeller Foundation.

[17] A S y s t e m a t i c A p p r o a c h to C h e m i c a l D N A S e q u e n c i n g b y S u b c l o n i n g in p G V 4 5 1 a n d D e r i v e d V e c t o r s 1 B y G U I D O VOLCKAERT

In the chemical degradation sequencing method of Maxam and Gilbert, 2 a DNA molecule, labeled at one end, is fragmented in a basespecific manner. The DNA is treated with a chemical reagent under conditions where only one nucleotide per chain is modified. Often this modified i Supported by the Belgian National Fund for Scientific Research (NFWO). The author is a Research Associate of this organization. 2 A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 74, 560 (1977).

METHODS IN ENZYMOLOGY, VOL. 155

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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base is prone to further disruption and the polynucleotide chain can be chemically cleaved at this position. If the reaction is base-specific and random, a set of degradation products of different lengths is produced. After fractionation according to chain length on a denaturing polyacrylamide gel, the labeled fragments are localized by autoradiography and the positions of the particular base (corresponding to the reaction specificity) along the chain can be noted in ascending order from the labeled end (either 5' or 3'). 2 Using an appropriate set of different reactions with different base specificities, run alongside each other on the sequencing gel, the complete nucleotide sequence can be "read" over a distance of several hundreds of nucleotides. The sequencing gel technology is common to all modern sequencing methods, and will not be dealt with here. The DNA fragments to be sequenced must be labeled at one end. Ordinarily, restriction fragments are used as substrates for that purpose and labeling can be obtained at either a 5' or 3' end. Since labeling usually occurs on both strands, a segregation step is needed so that subsequent chemical reactions are carried out with fragments labeled in one strand. Initially,2 selection of suitable fragments was based on a restriction cleavage map to determine the number of fragments to be sequenced. Subsequently, 3,4 a more random procedure was used by dispersed cutting and sequencing with different restriction enzymes until appropriate overlapping runs were obtained. These strategies involved several gel fractionations and D N A extractions. Another route to chemical sequencing consists of subcloning fragments in sequencing vectors. 5-9 This approach allows the use of restriction sites of the vector as targets for labeling and cleavage. Thus, the labeling procedure becomes a standard routine, independent of the physical map of the DNA to be sequenced. The restriction sites involved in the sequencing protocol are clustered in the vector close to one side of the insert. 5 Two unique sites are required; the site closer to the insert serves as the target at which the recombinant DNA is cleaved and radioactively labeled. The second restriction site (distal to the insert) is then used to cut off a small fragment (less than 30 bp) bearing one of the labels. No gel purification step is required since the small fragment will eventually cause 3 A. M. Maxam and W. Gilbert, this series, Vol. 65, p. 499. 4 j. G. Sutcliffe, Cold Spring Harbor Syrup. Quant. Biol. 43, 77 (1978). A. Frischauf, H. Garoff, and H. Lehrach, Nucleic Acids Res. 8, 5541 (1980). 6 U. Rtither, M. Kaenen, K. Otto, and B. Miiller-Hill, Nucleic Acids Res. 9, 4087 (1981). 7 p. Prentki and H. M. Krisch, Gene 17, 189 0982). s j. Vieira and J. Messing, Gene 19, 259 (1982). 9 G. Volckaert, E. De Vleeschouwer, H. B16cker, and R. Frank, Gene Anal. Tech. 1, 52 (1984).

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233

only a short ladder (superimposing on the ladder of the insert DNA) in the bottom part of the sequencing gel. 5 Bidirectional sequencing is also feasible if another set of unique sites is available at the opposite side of the insert, t° Large DNAs are reduced to a set of subclones which are sequenced and sorted by overlapping. This may be approached by "shotgun" subcloning of randomized fragments, 6,9 or in a systematic fashion by creating a series of progressively larger deletions 5,9 in the original DNA. Ideally, the gap between consecutive deletion borders is slightly less than the reading distance on a sequencing gel. Useful chemical sequencing vectors are pUR222, 6 pUR250,1° and the pUC 8 plasmid series, as well as other vectors which were not intentionally designed for this purpose, e.g., the pEMBL series. 11 Another class of chemical sequencing vectors are the pCSV plasmids. 9 These plasmids make single-end labeling of subcloned DNA extremely easy and rapid, because cleavage of the DNA and labeling (at a 3' end with Klenow polymerase) is reduced to a single, contiguous reaction. With these vectors, the secondary restriction cleavage is obsolete (see below). The pCSV vectors are derivatives of pBR322 which yield a reasonable amount of DNA per milliliter of a stationary culture of Escherichia coli cells. More sophisticated derivatives, however, have been developed which increase the DNA yield several fold. 12This improves the purity of the DNA, increases the sensitivity of the method, and facilitates simultaneous handling of many clones. The prototype of these novel vectors is pGV451; its properties and use in sequencing will be described here in detail. Other derivatives provide different (series of) cloning sites and are better suited for particular applications such as forced cloning and directional deletion. 13All of them exhibit the same replication mode as pGV451 and can be used following the same sequencing protocols. In the second part of this chapter the experimental procedures are described which have been developed and extensively used in combination with pGV451 and its derivatives. Miniprep DNA is used throughout the protocols. [35S]dNTP analogs are preferred for labeling as they result in sharper bands and increased resolution on the sequencing gel. 14 Moreover, prolonged storage of 35S-labeled chemical degradation products l0 U. Rtither, Nucleic Acids Res. 10, 5765 (1982). 11 L. Dente, G. Cesarini, and R. Cortese, Nucleic Acids Res. 11, 1645 (1983). ,2 K. Neesen and G. Volckaert, manuscript in preparation (1986). ,3 S. Henikoff, Gene 28, 351 (1984); see also S. Henikoff, this volume [12]. 14 M. D. Biggin, T. J. Gibson, and G. F. Hong, Proc. Natl. Acad. Sci. U.S.A. 80, 3963 (1983).

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does not reduce the quality of the sequencing gels significantly)5 The chemical procedures described in this chapter are adapted from the original protocols. 2,31 attempted to develop faster methods in two ways. First, several precipitation and/or evaporation steps were dropped without deleterious effects on the quality of the degradations. Second, the number of manual operations, such as mixing, opening and closing of tubes, spinning, additions, pipet adjustments, etc., was reduced by appropriate changes in sample volumes. With these adaptations, the chemical degradation reactions take no longer than about 90 min. Since plasmid isolation and labeling is done in less than 2 hr, the overall time Schedule (to gel loading) lasts no longer than 3.5 hr for a skilled and experienced sequencer. Biological Properties of p(3V451 pGV451 is a small, multicopy plasmid of 1579 bp. The exact copy number is difficult to measure since a stationary culture of E. coli bearing the plasmid contains threadlike (multicellular) structures rather than single cells. A minimal estimate is 1500 copies per cell. 12Although cloning of DNA fragments into pGV451 often reduces the copy number to various extents, yields of several micrograms (up to 10 /zg vector) DNA per milliliter are readily obtained. The physical and functional map of pGV451 is shown in Fig. 1. Figure 2 represents the complete nucleotide sequence, as well as a partial restriction cleavage list. Numbering of the map starts in the middle of a sequence of three deoxynucleotides,4,16 where RNADNA transition occurs during initiation of replication. Replication occurs in a clockwise direction. pGV451 carries the fl-lactamase gene promotor P317 (bla) of pBR322 in front of the (promoterless) chloramphenicol acetyltransferase gene (cat) of pBR325. TMThe cat gene, in turn, is followed by a 480-bp fragment of the origin of replication (ori) of pMB 1 (resembling closely the ori of ColE l).16 Thus, initiation of replication is dependent on transcription from the bla promoter. By the same token, the complete plasmid genome, except the region from n = 2 to about n = 290, is essential for survival of the plasmid 1~ I have occasionally rerun samples which were left in loading solutions at ambient temperature for 2 weeks, or in the refrigerator for several months. The quality of the corresponding sequencing gels had not changed visibly. Under the same conditions, 32p-labeled samples caused a significant appearance of secondary bands and smearing on the sequencing gels. 16 j. Tomizawa, H. Omori, and R. E. Bird, Proc. Natl. Acad. Sci. U.S.A. 74, 1865 (1977). 17 D. Stiiber and H. Bujard, Proc. Natl. Acad. Sci. U.S.A. 78, 167 (1981). Is F. Bolivar, Gene 4, 121 (1978).

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235

ori

SC

~, Pbie

1579 bp

b

$c

P

eat

ori

Bsmt ~THllll II I HINDlll

21

[

l

~LI ] SNAI

i

BANH1

I

1

g~Ll HII~II ACCI 1~II

t

11 1 1

PUUII ECORI NCOI

SCAI l-hClEll ,~qU3AI HINFI

2 1

[ [

1 1 1

I I

_~

~ [

1 1 4 3

HhEIII

?

hLUI

9

FIG. 1. Functional and physical map of pGV451. In (a), the c a t coding sequence is shown by an open bar and the fragment of the pMB1 replicon, by a filled bar. In (b), a computeraided drawing of a partial restriction map is represented. Enzyme names are at the left-hand side, whereas the number of cleavages by each enzyme is shown at the right. Pbla [in (a)] and P [in (b)] localize the fl-lactamase promoter. SC is the sequencing configuration.

in the cell. In the nonessential region, a series of unique restriction sites is located, flanked by HindIII and XmnI at the left-hand and right-hand sides, respectively. This segment is named "the sequencing configuration" and is described below. pGV451 confers chloramphenicol resistance to transformed E. coli

a

ACGCCAGCAAEGCGBCCCGA AGCTTATEGGTGACCETGAC TAAGTCBAGCCCAATTCCCG

GGGATCCGTC GACCTBCAGE CAAGCTGGGC

TCGACTCAGT EAGGGCGAT5 ATAAGCTGTC

GO

120

AAAC~TGABA ATTGAAGATCTTEGAAGATCTTCAATTCTTGAAGACSAAASGGCCTCGTS

180

ATACGCCTAT

2~0

TTTTATAGGT

TAATGTCATG

ATAATAATGG TTTCTTAGAC

GTCAGGTGGC

ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTAT TTTTCTAA~T ACATTCAAAT

300

ATGTAICCGC TCATGAGACAATAACCCTGATCGAGATTTTCAGGAGCTAAGGAAGCTAAA

380

ATGGAGAAAA AAATCACTGGATATACCACCGTTGATATATCCCAATGGCA TCGTAAAGAA

~20

CATTTTGAGG CATTTCAGTCAGTTGCTCAATGTACCT~TAACCAGACEGT TCAGCTGGAT

~80

ATTACBGCCT TTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCC5GCETTTATT

S~0

CACATTCTIG CCCGCCTGAT GAATGCTCAT CCGGAAITCC 5TATGGCAAT 5AAAGACGGT

500

GABCTGGTGA TATGBGATAGT G T T C A C C C T

GCAAACTGAA

650

G G C A G T T T C T ACACATATAT

720

ACGTTTTCAT EGETCTGGAG

IGTTACACCG T T T T C C A T G A

TGAATACCACG ~ C G A T T T C E

TCGCAAGATG TGGCGTGTTA CGSTSAAAAC CTGGCCTATT TCCCTAAAGG GTTTATTGAG

780

AATATGTTTT TCGTCTCAGC CAATCCCTGG GTGAGTTTCAEEAGTTYTGATTTAAACGTG

B~O

GCCAATATG5 ACAACTTCTT CGCCCCCGTT TTCACCATGG GCAAATATTA TACGCAAGGC

BOO

GACAAGGTGC TGATGECGCT GGCGAITCAG GTTCATCATG C~GTTTGTGA

BSO

TGGCTTCCAT

G T C G G C A G A A TGCTTAATGAA T T A C A A C A G T A C T G C G A T G A G T G G C A G G G C G G G G E G T A A

1020

TTTTTTTAAG GCAGTTATTG GTGCCCTTAA ACGCCTGGTG CTACGCCTGA ATAAGTGATA

1080

AIAAGCGGAI GAATGGCAGAAACTGGCTTCAGCAGAGEGC AGATACCAAAT A C T G T C C T T

ii~0

CTAGTGTAGC ~GTAGTTAGG CCACCACTTC AAGAACICTG TABCACCGCC TAEATACCTC

1200

GCTCTGCTAA TCCTGTTACC AGTGGCTGCI GCCAGTGGCG ATAAGTCGTG TCTTACCGGG

1260

TTGGACTCAA GAEGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCIGAAC GGGGGGTTCG

1320

TGCAEACAGC ECAGCTTGGA GCGAACGACC TACACCGAAC TGAGAIACET ACAGCGTGAG

1380

CATTGAGAAA GCGCCACGCT TCCCGAAGGG AGAAAGGEGG ACAGGTATCC GGTAAGCGGC

i~0

AGGGTEGGAA CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAAEGC~TG GTATCTTTAT

iSO0

A G T C C T G T C G G G I T T C G C C A C E T C I G A C T T G A G C G T C G A T TTTTGTGATGC T C G T C A G G G

1560

GGGEGGAGEC

TATGGAAAA

b E x c e r p t og the r e s t r i c t i o n r e c o g n i t i o n s i t e list of pGU~51

No s i t e s APAI ECOB NAEI SACII

ffor : AUAII ECOK NARI SFII

Unique s i t e s AAYII at BAMHI at HAEII at HINDIII at PUUII at

BCLI ECORV NBEI 5NABI

BGLI HPAI NDTI SPHI

ACCI BANI HGAI ~AEI BALI

at at at at at

BSSHII KPNI NRUI STUI

BSTXI MLUI NSII XBAI

AUAI BSTEII HGIAI NCOI SEAl

at at at at at

CLAI MSTI PUUI XH01

DRAIII MSTII SACI XMAIII

BALI ECORI HINCII PBTI 5MAI

at at at at at

: 228 52 1390 20 472

; ; ; ; ~

88 1050 1533 1151 88

; ; ; ; ;

57 2~ 1320 875 eBB

; ; ; ; ;

838 $7~ 58 75 57

; ; ; ; ;

P s e u d o - u n i q u e s i t e s (i.e. tuo s i t e s c l o s e to e a c h other) : BGLII positions at 13S 1~S ; Fragments i 0 ISB9 TTH1111 positions at 38 93 Fragments 58 182~ XMNI p o s i t i o n s ~t 13~ i~5 ; Fragments I0 155B

FIG. 2. (a) Complete nucleotide sequence of pGV451 and an excerpt of its restriction cleavage list. The ATG initiation codon and TAA termination triplet of the c a t gene are underscored. (b) A list of series of restriction enzyme sites absent in the vector and unique and pseudounique recognition sequences.

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237

cells. Intentionally, to keep the size of the plasmid as small as possible, no insertional inactivation marker has been provided. Dephosphorylation of cleaved vector, combined with purification on low-melting-point agarose, readily reduces background contamination of the clone bank with vector molecules. Principle of Sequencing with pGV451 and Derived Vectors End-specific labeling 19 with pCSV vectors, pGV451, and analogous plasmids 2° is based on filling-in of the protruding ends of pseudopalindromic restriction sites such as BstEII and Tthl 1lI. The recognition sites of these enzymes are interrupted hexameric sequences. (MstII or SauI, DraII, and RsrI, exhibit similar properties.) BstEII (G-G-T-N-A-C-C) cleavage generates the 5' overhangs G-T-CA-C and G-T-G-A-C or G-T-T-A-C and G-T-A-A-C, depending on whether a C/G or T/A internal base pair occurs. In the former case, filling-in polymerization with Klenow polymerase in the presence of dGTP, dTTP, and [a-35S]dCTP will label one end only, as illustrated here (label is denoted by an asterisk): 5'-G-G-T-C-A-C-C. . . . . . . 3'-C-C-A-G-T-G-G-

BstElI

>

-G --

pG-T-C-A-C-C-

.q-

-C-C-A-G-T-Gp

G-

Klenow dGTP,

-G-G-T-C* • . . . -C-C-A-G-T-Gp

polymerase dTTP,

q-

[ct-35S]dCTP

pG-T-C-A-C-C. . . . G-T-G-G-

Any DNA fragment, cloned at the right-hand side in proximity of this site, can be sequenced directly, provided that it does not contain a similar sequence itself (i.e., G-G-T-~-A-C-C is not labeled but interrupts reading of the sequencing gel at the position of occurrence; the presence of G-GT-C-A-C-C in the insert results in double labeling of the recombinant plasmid and thus precludes determination of the nucleotide sequence). Similarly, the TthlllI recognition sequence consists of two trimers ~9G. Volckaert, G. Winter, and C. Gaillard, "Advanced Molecular Genetics" (A. Ptihler and K. N. Timmis, eds.), p. 255 and ft. Springer-Verlag, Berlin, 1984. z0 G. Volckaert, A. Van Aerschot, K. Neesen, R. Frank, and H. B16cker, manuscript in preparation (1986)•

238

RAPID METHODSFOR DNA SEQUENCEANALYSIS

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separated by three (arbitrary) base pairs: G-A-C-N-N-N-G-T-C. Cleavage occurs symmetrically in front of the central base pair, so that a single 5' overhanging nucleotide remains at each end. In pGV451 (and in most of its related vectors), two T t h l l l I sites are present, each with a different central base pair. Hence, restriction cleavage with Tthl 11I can be drawn as follows: 5' -G-A-C-T-A-A-G-T-C . . . . . . . . . 3' -C-T-G-A-T-T-C-A-G

insert DNA

C-A-C-T-C-A-G-T-C. . . . . . . . .

C-T-G-A-G-T-C-A-G-

T

T

~lTthllI -G-A-C-T . . . . -C-T-G-A-T

+

A-A-G-T-C . . . . T-C-A-G

insert DNA

G-A-C-T C-A-G-T-C. . . . + . . . . C-T-G-A-G T-C-A-G-

Obviously, only one of the four ends can be labeled with a single radioactive dNTP precursor. With either labeled dTTP or dCTP, the insert is labeled and sequenced from one of its ends. Assuming that 300 nucleotides are " r e a d " from each end, this offers the possibility to sequence inserts of at least 550 bp long. E1 The set of restriction sites for subcloning and labeling in pGV451 and derived vectors is designated the "sequencing configuration." The sequencing configuration in pGV451 extends from position n = 20 to n = 153 and is represented in Fig. 3. Arrowheads show the direction of sequencing and mark the dNTP to be used as labeled precursor. For bidirectional sequencing of fragments cloned between the two Tthlll! sites, two approaches are feasible: one of them requires two labeling experiments on Tthl 1lI cleaved DNA with different radioactive precursors (dCTP and dTTP); the second approach involves two separate restriction digestions (BstEII and TthlllI), which both are labeled with dCTP. The choice may be dictated by the availability of the respective enzymes and labeled precursors to the experimenter, or by the incidental presence of a BstEII or TthlllI sequence in the insert. The statistical chance that sequencing would be impeded by the presence of both recognition sites within a fragment of 550 bp is negligible. 21 R e m e m b e r ,

h o w e v e r , t h a t it is a d v i s a b l e t o s e q u e n c e b o t h s t r a n d s c o m p l e t e l y .

ll 7] H~naIll

SstEII

CLONINGAND CHEMICALSEQUENCINGWITH pGV451 Tt__htll!

~ccl H~ncl I 5~al BamMI $~iI Pstl

TthlllI

239 Xmnl ~II

Xmnl B@~II

A~C~IAT~G~CCT@~TAA~TCGAG7CCA~CCEGB~GA~C~TCGA~TGC~CCA~CTGGB~T~ACTCA~TCAGBBC~AT~A~B~T~T~AAC~TBAGAATT~AAB~TCTTC~AA~AT~TTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TTc6~ATAGcc~zTGBBA~TB~AG~TZ6~B~aa6GG~cc~GG~G~6B~cGI~GG~T~G~EccGA~BAGTc~cccB~AcTAT~B~TrGTACICZTA~CTTCT~B~aBCTTCTAGAAG

FIG. 3. Sequencing configuration in pGV451. The DNA sequence from n = 20 to n = 153 is shown. BstEII and Tth 11 lI are labeling sites. Dots on ends of arrows mark the nucleotide that transfers tile radioactive label onto the DNA and the corresponding arrow indicates the direction of sequence reading. HindlII, BglII, and XmnI are cloning sites for unidirectional sequencing only.

Materials

and Reagents

Plasmids and Bacterial Strains pGV451 has been circulated to many laboratories during the past years and can be obtained from the author. If you need cloning sites other than the ones present in pGV451, other derivatives will be sent if you specify your requirements, pGV462 and 463 are two derivatives with multiple cloning sites adapted to progressive deletion experiments with exonuclease III. 13 All procedures of the sequencing method have been extensively tested and optimized with JM8322 as host for transformation and source of DNA. Other strains have been occasionally used successfully: DS41023 allows the study of cloned DNA fragments in minicells; DNA purified from GM11924 does not contain 5-methylcytosine in C-C-~-G-G sequences so that no gaps occur in the eventual sequencing ladders; HB101, 25 which I used originally 9 with the pCSV and early pGV sequencing vectors, can be used to prepare vector DNA, but is not recommended for cloning because many pGV451 recombinants are unstable in this strain. All strains are grown in LB medium (1% Bacto tryptone, 0.5% Bacto yeast extract, and 0.5% NaCI). To prepare 1 ml of competent cells, a culture is grown to late logarithmic growth phase (about 2 x 108 cells/ml). 100 ml of the culture is centrifuged at 5000 rpm in a Sorvall centrifuge (rotor GS3, Dupont) (about 3600 g) for 5 rain. The cell pellet is chilled and resuspended in 10 ml ice-cold 100 mM CaCI2. The cells are kept on ice for 30 min and spun down again as before. The pellet is finally resuspended in 1 ml ice-cold 100 mM CaC12. To prepare a larger stock of competent cells, adapt the volumes accord22 C. Yanisch-Perron, J. Vieira, and J. Messing, Gene 33, 103 (1985). 23 E. Dougan and D. Sherratt, Mol. Gen. Genet. 151, 151 (1977). 24 M. G. Marinus and N. R. Morris, J. Bacteriol. 114, 1143 (1973). z5 A. Dugaiczyk, W. Boyer, and H. M. Goodman, J. Mol. Biol, 96, 171 (1975),

240

RAPID METHODS FOR D N A SEQUENCE ANALYSIS

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ingly. If the cells are not used for transformation within 72 hr, add glycerol to a final concentration of 15% and store in aliquots at -70 °.

Enzymes BstEII is obtained from Boehringer Mannheim, TthlllI from Amersham International and New England Biolabs. Other restriction enzymes are purchased from different suppliers, as well as Klenow (large fragment) DNA polymerase I. The quality of the latter enzyme is less demanding than in dideoxy sequencing. Bacterial alkaline phosphatase (BAPF in ammonium sulfate suspension) from Worthington Biochemical Corp. is spun down for 2 min in a microcentrifuge and redissolved in water at a concentration of 1 mg/ml. DNA ligase is obtained from Boehringer Mannheim; lysozyme is from Sigma. Chemicals and Buffers for Cloning and Labeling This is an informal list only. Products from other sources may also be acceptable after proper testing: rATP, unlabeled dNTP, DTT (dithiothreitol), BSA (bovine serum albumin): Boehringer Mannheim. [35S]dCTP: Amersham International. [35S]dTTP, dATP, and dGTP: Dupont (New England Nuclear). CDTA (trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, monohydrate): Sigma. Low-melting-temperature agarose: Gibco (BRL). Sephadex G-50, medium: Pharmacia: swell in 0.1 mM NaC1. Sucrose: UCB, Belgium. SDS (sodium dodecyl sulfate): Fluka AG. EDTA disodium (Titriplex ®III), n-butanol, MgCI2, CaCl2, sodium and potassium acetate, Triton X-100, PEG 6000 (polyethylene glycol): Merck. NaC1, Tris, phenol: Janssen Chimica, Belgium. Phenol is equilibrated with 1 M Tris. HCI (pH 8) and stored at -20 °. A working solution is kept at 4° in the dark for several weeks. Distillation of this product is not necessary. 10× ligation buffer: 200 mM Tris. HCI and 100 mg MgC12(pH 7.6). STCT buffer: 5% Triton X-100, 8% sucrose, 50 mM CDTA, and 50 mM Tris. HCI (pH 8.0). CTLM buffer: 0.2% Triton X-100, 25 mM CDTA, 25 mM EDTA, and 25 mM Tris. HCI (pH 8.0).

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CLONINGAND CHEMICALSEQUENCING WITHpGV451

241

BstEII cleavage buffer: 0.3 M NaC1, 12 mM MgC12, 12 mM 2-mercaptoethanol, 100/~g/ml BSA, and 12 mM Tris. HCI (pH 9.0).

BstEII labeling buffer: 60/~M dGTP, 60/xM dTTP, 5 mM DTT, l0 mM MgCI2, and 30 mM Tris. HC1 (pH 7.6).

TthlllI cleavage buffer: 0.1 M NaC1, 16 mM MgC12, l0 mM DTT, and 50 mM Tris. HC1 (pH 7.4). TthlllI labeling buffer: 10 mM DTT, l0 mM MgClz, 50 mM Tris. HC1 (pH 7.6).

Buffers and Reagents for Sequencing Analytical-grade chemicals from different suppliers have been used for chemical degradation sequencing. The most important concern is proper storage and handling of the critical products. Deterioration of hydrazine is the major problem encountered. Keep hydrazine in the original bottle in a refrigerator. (Never aliquot it into plastic vials !) Take it out of the refrigerator only at the moment you need it and put it back immediately. Using these precautions I am still able to use the same bottle of hydrazine I started 5 years ago. It is advisable to apply the same procedure with dimethyl sulfate and piperidine. The safe handling of hazardous reagents has already been discussed thoroughly in this series. 3 The following solutions are stored at 4°: precipitation mix 1:0.3 M ammonium acetate and 30/xg/ml carrier RNA precipitation mix 2:1.5 M ammonium acetate and 60/zg/ml cartier RNA AG-reagent: 2% formic acid (pH 2), with pyridine 1.2 M NaOH and I mM EDTA 5 M NaC1 is stored at ambient temperature.

Disposable Materials Most of the manipulations with DNA are carried out in 1.5-ml plastic microcentrifuge tubes. Eppendorf (Netheler+Hinz, GmbH, Hamburg, Federal Republic of Germany) and Treff tubes (Treff AG, Degersheim, Switzerland) are satisfactory. Some other brands probably work well. Note, however, that there are many tubes of poor quality to which DNA tends to stick. Both companies sell tubes in five different colors (see Methods). Unless otherwise indicated, all sampling, dispensing, and transfer operations of DNA solutions, reagents, solvents, and superna-

242

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tants are done with pipet tips on pushbutton micropipets. Gilson Medical Electronics (France) and Treff are suitable sources. Again, caution should be taken in selecting other kinds of pipet tips. Disposable glass tubes of 75 × 8 mm (internal diameter) are used as recipients for quick desalting. These glass tubes constitute the lower part of a small device for purification of DNA samples less than 250 ~1 in volume. The upper part is a Gilson P1000 micropipet tip which fits in the glass tube, but rests with its wider end on the rim of the tube. A siliconized glass bead (about 3 mm diameter) is trapped in the tip stricture and 1.5 ml swollen Sephadex G-50 (medium size) is poured onto it. The Sephadex G-50 does not leak 26 past the glass bead under appropriate conditions. The Sephadex G-50 is drained dry by a 2-min centrifugation at 2200 rpm in a Labofuge GL (Heraeus Christ, rotor 2150). Subsequently, the DNA sample is layered on top of the Sephadex G-50 and spun through at 1800 rpm. With other clinical centrifuges, spinning conditions must be determined by some trial and error. It is important to use a "swinging bucket" rotor. Recovery of the DNA is 80-100%; the volume of the eluate is usually about two-thirds of the original sample, but even when smaller volumes are recovered, the DNA is recollected completely in the eluate. I refer to this device and procedure in the protocols as "filtration through a Sephadex G-50 tip."

Equipment 302-nm UV light transilluminator. Sonicator with cup horn attachment, e.g., model W-375 of Heat Systems Inc. A well-ventilated fume hood is absolutely required to carry out the chemical degradation reactions with hydrazine and dimethyl sulfate. Keep a bottle containing FeC13 fragments in this hood to dispense waste produced by these reactions. Dimethyl sulfate is destroyed by the piperidine reaction (see Methods). Volatile reagents such as piperidine can be removed from the samples by lyophilization or evaporation in a stream of air. The Speed-Vac concentrator (Savant Instruments Inc., New York) is convenient to dry the sample by a combination of heat (about 45 °) and vacuum. This instrument is used in the protocols described here and is designated vacuum centrifuge. A vacuum of at least 100 mTorr should be reached. An Eppendorf thermostat block (or equivalent device) is used for heating of samples at 95 ° and 37° . Racks (No. 3831) which fit on this Fine and superfine grades are not suitable.

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heating block are sold by the same distributor. The racks are also transferable to the Eppendorf mixer (No. 5432), which is a valuable help if many clones are to be sequenced simultaneously. Method

Cloning Preparation ofDNAfor Subcloning. Several methods have been used to subclone large DNAs in pGV451. 1. Cloning of sticky-end restriction fragments in the appropriate site of the sequencing configuration: BamHI, SalI, AccI, PstI, BgllI, and HindlII. Blunt-end fragments can be cloned in SmaI, HinclI, or XmnI. This is done by standard procedures. 2. Subcloning of dispersed fragments from blunt-end restriction digestions: Cleave DNA with AluI, DpnI, MnlI, HaelII, FnuDII, and RsaI in 2-/~g portions in separate tubes. (Other enzymes may also be included in this list, e.g., Hinfl, TaqI, MspI, DdeI, but these fragments must be treated with Klenow polymerase and dNTPs prior to the sizing procedure.) After incubation, heat the digests at 70° for 10 min and transfer them to a single tube containing a volume of equilibrated phenol that equals the pooled fractions. Extract the aqueous phase, discard the phenol, add sodium acetate to 0.3 M, and precipitate with 2.5 volumes of ethanol. Redissolve the DNA in 10/.d water and add 10/~1 14% PEG 6000/1 M NaCI. Keep 30 min at 0°. Spin down the precipitate for 10 min and remove the supernatant completely. Repeat this procedure twice. Finally, redissolve the pellet in 10/zl water, add 8/~1 14% PEG 6000/1 M NaC1, and chill at 0° for at least 3 hr. Then collect the precipitate again by centrifugation as above and redissolve at an estimated concentration of 5 ng/~l. It is advisable to save the last supernatant and to check one-tenth of the pellet on an agarose gel. 3. Randomization of the DNA by shearing: Circularize the DNA to be sequenced by a standard ligation reaction. Extract with phenol and precipitate the DNA. Dissolve the DNA in 35/zl water, add 5/zl 1 M NaC1, and I0/~1 200 mM Tris. HCI/50 mM MgClz (pH 7.6) in a microfuge tube. Attach the tube in the cup horn device of the sonicator and give four bursts of 40 sec each. Sonication of DNA produces a variety of different ends (blunt, 5' and 3' extensions, 5' and 3' terminal phosphates, and OH ends). Hence, more DNA than usual is needed for subcloning. The clon-

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ing yield can be improved by treatment of the sonicated DNA with Klenow polymerase and dNTPs: add 4/zl of each of 1 mM dNTP, 10 ~1 DTT, 20/xl water, and 2 units Klenow polymerase. Incubate several hours at 16°. Add 20/.d 0. I% SDS/50 mM EDTA. Extract with 50/~1 equilibrated phenol and filter through a Sephadex G-50 tip. If the eluate is larger than 20/~1, concentrate it in the vacuum centrifuge. Subsequently, add 5/zl of 60% sucrose/l% bromphenol blue and load the sample on a 2% low-melting-temperature agarose gel. Run reference DNA fragments alongside the sheared DNA. Finally, stain the gel with 1/~g/ml ethidium bromide and excise the fraction which is 400 to 600 bp in size under UV light of 302 nm. Elute the DNA fragments by the same procedure described for extraction of vector DNA in the next section. Redissolve the DNA at an estimated concentration of 20 ng/ml. 4. Directional progressive deletion of DNA: This procedure is described in detail in chapter [12] of this volume. 13

Cleaoage and Purification of pGV451 for Subcloning. The following procedure will provide cleaved vector for cloning of fragments generated according to steps 1, 2, or 3, of the previous section. Miniprep pGV451 DNA is suitable and is isolated according to the same procedure described in a further section of this chapter. Digest 10/xg pGV451 DNA with the appropriate restriction enzyme in 50/zl of the buffer specified by the supplier of the enzyme (but avoid using ammonium salts because they inhibit subsequent dephosphorylation). For cloning of blunt-end fragments, Sinai is ordinarily used. Add 2/zl 1.0 M Tris. HC1 (pH 8.3) and 3/.d bacterial alkaline phosphatase. Incubate for 15 min at 37°, then for 10 min at 56°. Add 10/zl 60% sucrose/l% bromphenol blue. Run the digest on a 2% low-melting-temperature agarose gel (with 0.5/~g uncleaved pGV451 in an adjacent slot as a reference). Stain with 1/zg/ml ethidium bromide, transfer the gel onto the transilluminator, and excise the band containing linear vector. Transfer the gel fragment to a microfuge tube. Heat the tube at 65° until the gel is melted (at least 5 rain). In the meantime, distribute 400-/zl aliquots of equilibrated phenol (pH 8.0) to each of three microfuge tubes and arrange them at 37° in the thermostat block. Add 350/xl 50 mM Tris. HC1 (pH 8.0)/0.5 mM EDTA (prewarmed at 65°) and 600/zl neutralized phenol (37°) to the melted gel slice. Extract, spin 1 min in the microfuge, and transfer the aqueous phase to the first tube in the thermostat. Re-extract the phenol with 100/zl water and combine both water phases. Repeat the extraction procedure three more times

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with 300/zl phenol (but without re-extraction of the phenol). Extract the final water phase with n-butanol (which forms the upper phase). Repeat several times until the volume is reduced to about 300/zl (usually two or three times). Add 100/zl 1 M sodium acetate (pH 7.5)/0.3 mM EDTA. Add 1200/.d ethanol, mix, chill at - 7 0 ° for 10 min (dry ice-ethanol bath), and spin down precipitate for 10 min. Remove the supernatant, add 800/xl 80% ethanol, vortex, centrifuge 1 min, discard supernatant, and repeat rinse once more with 80% ethanol. Dry the pellet in the vacuum centrifuge. Redissolve the vector in water at a concentration of 50 ng//xl and store in aliquots to avoid repeated freezing and thawing. Ligation and Transformation. Prepare five microfuge tubes by combining in each 2/xl cleaved vector, 5/xl 10x ligation buffer, 5/zl 100 mM DTT, and 5/xl 10 mM rATP. Add 33/.d water to tubes 1 and 2. To tubes 3, 4, and 5, transfer 33 /zl of undiluted, 3x diluted, and 9× diluted DNA fragments, respectively. This represents a molar ratio of fragments to vector of 6, 2, and 0.7, respectively, with enzyme-digested DNA. With sonicated DNA, these ratios are four times higher. Add 2 units ligase to tubes 2, 3, 4, and 5, and incubate at ambient temperature for 1 hr. With sonicated DNA, use 5 units ligase instead of 2. Dispense 200/xl competent cells in a standard 10-ml tube for each transformation. Competent cells stored at -70 ° are thawed slowly on wet ice. Add the DNA sample (0°) to the cells. Leave on ice for 5 min. Heat shock the cells by keeping the tubes 30 sec. at 37° and 30 sec at 0°. Repeat this cycle five times. 9 Place the tubes again on ice for 10 min, then add 2 ml LB broth and shake at 37° for about 45 min. In the meantime, melt top agar and dispense 4-ml samples in l0 tubes at 44 °. Add 10 ~1 of 60 mg/ml chloramphenicol (dissolved in ethanol or ethylene glycol) to each of the tubes. Add 200/~I of the transformation mixes 3, 4, and 5 to tubes with agar, mix briefly, and pour on LB plates containing 25/zg/ml chloramphenicol. For the rest of all transformation mixes (about 2 ml each), mix the contents of a tube with agar and plate out as before. Invert the plates after solidification of the agar and incubate at 37° overnight.

Isolation and Labeling of Subclone DNA Preparation of Subclone DNA. Toothpick subclones in 1.5 ml LB broth with 25/xg/ml chloramphenicol and grow overnight. Pellet the cells in a microfuge tube and discard the supernatant. (Centrifuge no longer than 30 sec as the bacterial pellet may be difficult to resuspend.)

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Use one of the following lysis protocols: (a) Recipe adapted from Holmes and Quigley27: Resuspend bacteria in 120/zl STCT buffer. Add 10 /zl of 20 mg/ml lysozyme in 25 mM Tris. HCI and 25 mM CDTA (pH 8.0). Mix by gently vortexing. Leave for EXACTLY 1 min at 100° in a boiling water bath. (b) Modified Triton X-100 lysis procedureg: Resuspend bacteria in 100/xl 10% sucrose and 50 mM Tris. HCI (pH 8.0). Add 20/zl of 20 mg/ml lysozyme in 25 mM Tris. HCI and 25 mM CDTA (pH 8.0). Mix by gently vortexing. Add 120/zl CTLM. Lysis occurs almost instantly. For either of the above methods, continue as follows: clear the lysate by centrifugation for 10 min in a microfuge or in a Sorvall centrifuge (rotor SS34, adaptor 381) at 20,000 rpm (about 24,000 g). Transfer the supernatant to a microfuge tube containing 120/zl equilibrated phenol. Extract by vortexing, centrifuge for 1 min. in a microfuge, and filter the aqueous phase through a Sephadex G-50 tip. Transfer the eluate to another microfuge tube and add 2.5 volumes of 0.1 M potassium acetate (pH 6) in ethanol. 28 Mix thoroughly. A precipitate forms immediately, which is collected by 1-min centrifugation in a microfuge. Remove the supernatant completely and dry the pellet in the vacuum centrifuge. Cleavage and Labeling 1. BstEII. Dissolve the pellet in 10/zl water. Add 10/zl BstEII cleavage buffer and 10-20 units BstEII. Keep 15-30 min at 60°. Add 60 /xl BstEII labeling buffer, 0.5 /xl a-35S-labeled dCTP, and 1 unit Klenow polymerase, in this order. Incubate 5 min at room temperature. Add 20/zl 0.2 M CDTA and 0.2% SDS, and 100/xl 14% PEG 6000 and 2 M NaC1. Mix and chill 20 min at 0°. 2. TthlllI. Dissolve the pellet in 30/zl in water. Add 30/zl TthlllI cleavage buffer and 10-20 units TthlllI. Keep 30 min at 65°. Add 10/zl 1 mM dATP and 130/zl Tthl 11I labeling buffer. Distribute two 100~1 portions in two microfuge tubes and add 1/zl a-35S-labeled dCTP to one of the tubes and 1/zl ot-35S-labeled dTTP to the other. Add 1 unit Klenow polymerase to each tube. Incubate for 5 min at room temperature. Add 50/xl 0.2 M CDTA, 0.2% SDS, and 150/xl 21% PEG 6000 and 3 M NaCI. Mix and chill 20 min at 0°. With either of the above labeling procedures, continue as follows: Spin 10 min in a microfuge. Remove the supernatant completely. Wash the pellet with an excess of 80% ethanol. (The DNA must be salt free to warrant a proper T + C chemical degradation. If weak T bands are ob27 D. S. Holmes and M. Quigley, Anal. Biochem. 114, 193 (1981). 28 The solution is prepared by mixing 3 ml of 3 M potassium acetate (pH 6.0) with 97 ml of ethanol.

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tained, an additional ethanol wash may be required.) Heat the tube for 2 min in the Eppendorf thermostat at 95 ° to evaporate traces of ethanol. Chemical Degradation Phase 1. Redissolve the pellet in at least 14 /zl water. Use larger volumes if you want to save some labeled DNA for other purposes, e.g., hybridization experiments with Tthl 1lI-cleaved DNA or repetition of one (or more) of the reactions. Five microfuge tubes are needed for each DNA. The tubes are conveniently arranged in a rack that fits onto the thermostat heater and the mixer. For centrifugation, tubes must be transferred manually to the microcentrifuge. Labeling of the tubes according to reaction specificity can be avoided by using colored tubes. Distribute each labeled subclone DNA in colored tubes as follows:

Reaction specificity G: 2/zl A + G: 3/xl A + C: 3/xl

T + C: 4/xl C: 2/zl

Color code Red (pink) White Blue Green Yellow

Put the A + G tubes in the thermostat at 95° until complete evaporation has occurred (about 5 min). Phase 2. Prepare fresh solutions as follows. The volumes described here are for eight clones, but are reduced or increased for fewer or more clones if appropriate. Pip 1:1000/zl water + 120/zl piperidine (dispense the piperidine into the water at the bottom of the tube). Pip 2 : 3 0 0 / x l water + 50/zl piperidine. G-buffer: 500/zl water + 15/xl Pip 1. G-reagent: 515 /~1 G-buffer + 1 /~1 dimethylsulfate (manipulations done in a fume hood). AC-reagent: 70/xl 1.2 M NaOH and 1 mM EDTA + 100/zl ethanol. The C-reagent and TC-reagent are prepared in a fume hood. Put two microfuge or glass tubes in an ice-water bath, labeled C and TC, respectively. Add 200/zl of 5 M NaCI to the C tube and 150/zl water to the TC tube. Leave to chill for 1 min. Dispense 300/xl hydrazine in each tube. Do not mix by vortexing but leave another minute on ice. Then vortex gently to mix and chill again for l min. The hydrazine dilutions are now ready and must be kept on ice till use.

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Phase-3.

Reactions are done in open tubes unless otherwise indicated. All reagents are added from the wall of the tube without unusual care so that the solutions mix slowly by themselves. Do not mix by any other means unless explicitly mentioned. If no temperature is mentioned, ambient temperature is meant. Chilling indicates the transfer of the rack containing the tubes into a dry ice-ethanol bath at - 7 0 °. Heating at 95 ° is always done in the Eppendorf thermostat; when heating over longer periods, collect any condensed reagent at the bottom of the tube by swinging the rack jerkily with your hand every 10 min. Cover the rack with a plexiglass plate to prevent popping of the lids. Spinning means centrifugation in a microfuge. Degradation at G: Add 5/~1 G-reagent. After 2 min, add 30/xl Pip 1, close the tubes, heat 25 min at 95°, and dry down in the vacuum centrifuge. Degradation at A + G: Add 5/~1 AG-reagent. Keep tubes 10 min at 37°. Add 30 t~l Pip 1, close the tubes, heat 25 min at 95°, and dry down in the vacuum centrifuge. Degradation at A + C: Add 17 ~1 AC-reagent. Close the tubes and heat at 95°. After 5 min, open the tubes and add 35 tzl Pip 2; close tubes again and leave them another 25 min at 95°. Add 100/~1 precipitation mix 2 and 500 t~l ethanol. Close tubes and mix. Chill 5 min, spin down 5 min, and discard supernatant. Degradation at T + C and C, respectively: Add 40/~1TC- or C-reagent to the respective tubes (note: the reagents are at 0°, but not the tubes containing the DNA). After 5 min, add 620 ~1 ice-cold ethanol and 220 tzl precipitation mix 1, in that order. Close the tubes, cover the rack with a plastic plate of the same size, and mix by inverting the rack 5 times. Chill the tubes immediately for 5 min (or longer), spin tubes 5 min, and discard the supernatant in a bottle containing fragments of FeCI3 .z Add 400/xl 75% ethanol, close the tubes, mix briefly by inverting, and spin tubes again for 2 min. Discard the supernatant. Transfer the tubes onto the thermostat at 95 ° and after 2 min, add 30 tzl Pip 1 to each tube. Close the tubes and keep them 25 min at 95°. Evaporate the piperidine in the vacuum centrifuge. Continue with all tubes as follows: wash the residue with 190 txl ethanol. Close the tubes, spin 2 min, and remove the supernatant. Heat all open tubes for 2 min at 95 °. The residue is redissolved in a solution of 0.3% xylene cyanole and

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0.3% bromphenol blue in formamide, usually 2 to 10/zl. From 1- to 2-/zl aliquots of each sample are loaded per sequencing gel. 14,29,30 Comments

Miniprep DNA of good quality in sufficient amounts is central to the sequencing method described here; 1 ml of cells yields enough pGV451derived clone DNA for several sequencing runs. Not every E. coli strain, however, is suitable for this purpose. Strains other than the ones mentioned above should be carefully tested first. Initially, it is advisable to estimate the yield of the isolation procedure by running a portion of the DNA on a 2% agarose gel. This may, however, be rather difficult because pGV451 and recombinant clones are prone to multimerization. The same problem is encountered when the size of inserts is analyzed by the same method. A solution to this problem is to linearize the DNA with an enzyme that cleaves the plasmid once (e.g., BstEII). Two protocols have been used successfully to prepare DNA for sequencing. Attempts to adopt the alkaline lysis procedure 31 often failed to give good sequencing ladders. 32 Short filling-in reactions with Klenow polymerase give better results than prolonged incubations with smaller amounts of enzymes. It is also essential that at least one of the four dNTPs is absent from the labeling mixtures. The PEG-induced precipitation concentrates the DNA and removes excess [35S]dNTP and any other small, labeled fragments, which sometimes accumulate in variable amounts. 9 RNA does not interfere with any of the cleavage, labeling, or degradation reactions, although it participates in some of them. Therefore, RNase treatment is not recommended at any stage of the method. As a consequence, carrier RNA is not strictly required in the precipitation mixes, unless the miniprep DNA has been purposely treated with RNase. Chemical sequencing with 35S-labeled DNA has the same advantages observed in dideoxy sequencing. ~4It requires, however, that the gels are washed and dried prior to exposure to an X-ray film. Overnight exposure is usually sufficient. The pGV vectors are also useful tools for other applications than chemical sequencing: the rapid restriction mapping method of Smith and 29 F. 30 H. 31 H. 32 A.

Sanger and A. R. Coulson, FEBS Left. 87, 107 (1978). Garoff and W. Ansorge, Anal Biochem. 115, 450 (1981). C. Birnboim and J. Doly, Nucleic Acids Res. 7, 1513 (1979). Meyerhans, personal communication and unpublished results (1985).

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Birnstie133 can be combined easily with the single-end labeling procedure. The end-labeling procedure can also be used to prepare insert- or strandspecific probes since cleavage with TthlllI excises the inserted DNA from the vector, and neither dCTP nor dTTP will label the vector fragment. Such probes can be used for mRNA mapping purposes and for rapid colony hybridizations to other members of a clone bank. The latter procedure provides another strategy to systematic sequencing by "walking" along the parental DNA from one clone to the other in a subclone bank. This is readily done by saving a portion of the labeled DNA when dispersing aliquots for the chemical degradation reactions. Other pGV derivatives are better suited for this application than pGV451 since the two Tthl 11I sites of pGV451 are separated by 55 bp. This fragment might cross-hybridize under less than stringent conditions. 33 H. O. Smith and M. L. Birnstiel, Nucleic Acids Res. 3, 2387 (1976).

[18] D i r e c t T r a n s f e r E l e c t r o p h o r e s i s U s e d for D N A S e q u e n c i n g By FRITZ M. POnL and STEPHAN BECK Introduction The separation of nucleic acid molecules differing in length by single nucleotides on denaturing polyacrylamide gels with high-resolution electrophoresis, together with methods for obtaining sets of molecules ending at particular bases, have revolutionized our knowledge of the chemical structure of DNA and RNA. The sequencing reactions using chemical degradation I or template-directed enzymatic synthesis with chain-terminating nucleotides2 are being constantly improved and simplified.3-5 Since the maximal number of nucleotides that can be read correctly in a single electrophoresis run is of importance for the economy of sequencing projects, considerable efforts have also been devoted to improving A. M. Maxam and W. Gilbert, this series, Vol. 65, p. 499. 2 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977). 3 j. Hindley and R. Staden, " D N A Sequencing." Elsevier, Amsterdam, 1983. 4 A. Rosenthal, R. Jung, and H.-D. Hunger, this volume [20]. 5 A. T. Bankier, K. W. Weston, and B. G. Barrell, this volume [7].

METHODS IN ENZYMOLOGY,VOL. 155

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