Gene synthesis

Double stranded DNA (ds-DNA) can be prepared by preparing a hybrid DNA containing a single stranded portion and a double stranded portion and carrying out in vivo gap repair on the hybrid DNA. The hybrid DNA is prepared by synthesizing a single strand of DNA and introducing the single strand into double stranded DNA. This method of synthesis can be used to make synthetic genes, including synthetic DNA coding for the TAT protein of HIV-I, and incorporates useful restriction sites. Also included are flanking restriction sites to simplify the incorporation of the gene into any desired expression system.

This invention relates to a method of synthesising double stranded DNA 
(ds-DNA), and to ds-DNA produced by such a method. ds-DNAs produceable by 
a method in accordance with the invention include synthetic genes coding 
for the transactivator of human immunodeficiency virus, to which the 
invention also relates. 
Examples of ds-DNA and in particular total gene synthesis are becoming 
increasingly common as the reliability of oligonucleotide synthesis and 
the efficiency of assembly methods continue to improve. Gene synthesis is 
now an invaluable tool for the molecular biologist because of the total 
control it affords over restriction sites, codon usage and subsequent 
genetic manipulation and expression of the gene. This is particularly true 
where the gene is refractory to manipulation because of a lack of useful 
restriction sites or because it is derived from a spliced RNA, as is true 
in the case of the transactivator (TAT) protein of HIV1. 
Methods of oligonucleotide synthesis have been reviewed extensively 
elsewhere (Gait, M. J. (1984) In M. J. Gait (ed), Oligonucleotide 
Synthesis: a Practical Approach, IRL Press, Oxford pp. 1-22). A number of 
different methods for the assembly of oligonucleotides have been described 
which divide into two main groups. In the first pioneered by Khorana and 
co-workers (Khorana, H. G. (1979) Science 203, 614-625), both strands of 
the desired sequence are divided such that adjacent pairs of complementary 
oligomers possess short (4-7 base) cohesive ends. The oligomers are then 
synthesised, kinased and annealed in pairs prior to ligation in a duplex 
corresponding to the intact gene. The ends of the gene are also endowed 
with cohesive ends to allow subsequent cloning of the gene in an 
appropriate vector. A recent development has been the successful solid 
phase assembly of a gene for cow colostrum trypsin inhibitor (Hostomsky, 
Z., Smrt, J., Arnold, L., Tocik, Z. and Paces, V. (1987) Nucelic Acids 
Research 15, 489-4856). The essential feature of these approaches is that 
both strands of the duplex are synthesised in their entirety. 
The second strategy is based on the use of longer oligomers that share a 
complementary 3, end (Rossi, J. J., Kierzek, R., Huang, T., Walker, P. A. 
and Itakura, K. (1982) J. Biol. Chem. 257, 9226). Annealing a pair of such 
oligomers results in a short duplex region with two long single stranded 
extensions. Treating this partial duplex with Klenow fragment of DNA 
polymerase I in the presence of all four dNTP's results in the conversion 
of this structure to a complete duplex with blunt ends. This method has 
been successfully applied to the synthesis of a gene for Eglin C (Rink, 
H., Liersch, M., Sieber, P. and Meyer, F. (1984) Nucleic Acids Research 
12, 6369-6387) and could in theory be extended to the construction of 
larger genes. It is attractive in that it reduces the amount of 
oligonucleotide synthesis required, but it has its drawbacks in that 
rearrangements and deletions are not uncommon. 
A method of synthesising lengths of ds-DNA, particularly synthetic genes, 
has now been developed which reduces the amount of oligonucleotide 
synthesis required in the Khorana method but which does not have all the 
drawbacks of the Klenow fragment method. 
According to a first aspect of the invention, there is provided a method of 
synthesising double stranded DNA, the method comprising preparing hybrid 
DNA containing a single stranded portion and a double stranded portion and 
carrying out in vivo gap repair on the hybrid DNA. 
The single stranded portion may comprise at least 10, 20, 50 or 100 
nucleotides. 
The hybrid DNA (which may be a vector such as a plasmid) may be prepared by 
synthesising a single strand of DNA and introducing the single strand into 
double stranded DNA. 
The single strand can conveniently be provided with double stranded ends 
and subsequently introduced into a double stranded vector. The double 
stranded ends of the single strand may be sticky, to correspond with 
restriction endonuclease cuts (natural or synthesised) in the ds-DNA, and 
will therefore for preference be different from one another. If a standard 
pair of sticky ends is used (for example HindIII and EcoRI), a "cassette" 
system is developed, whereby cassettes of various ss-DNAs can be inserted 
into, for example, a common vector system. 
The hybrid DNA may comprise at least two single stranded regions, the or 
each of the single stranded regions being separated by double stranded 
DNA. This embodiment has two advantages. First, the ds-DNA separator is 
useful in that it can be used as a sequencing primer. Secondly, the ds-DNA 
separator can form a means to link two separately synthesised ss-DNAs 
together if it is not feasible or convenient to synthesise the two as one. 
Preferred ds-DNA synthesised by a method in accordance with the first 
aspect of the invention includes a number of genes, particularly the tat 
gene of the Human Immunodeficiency Virus which encodes the transactivator 
TAT. 
In addition to the three principle structural genes gag, pol and env the 
Human Immunodeficiency Virus (HIV1) possesses a number of shorter open 
readinig frames with less well defined functions. The tat gene is composed 
of two exons and encodes the transactivator TAT, a regulatory protein 
involved in the activation of the HIV long terminal repeat (LTR). TAT is 
believed to exert its major effect on HIV gene expression by acting as an 
anti-terminator. Recent studies suggest that TAT interacts with a site in 
the transcribed R region of the LTR to prevent the premature termination 
of the RNA before it reaches the viral genes. Thus transcription beyond 
base 59 of the LTR derived transcript occurs only in the presence of TAT. 
The site at which TAT acts has been defined by deletion analysis and is 
known as TAR (for trans-acting response element). It is not yet known 
whether TAT exerts its effect via the genomic TAR or an mRNA structural 
motif encoded by TAR but it is becoming clear that regulation of 
expression from the 5' LTR involves the interaction of a number of other 
transcription factors in addition to TAT. 
The complete nucleotide sequence of a number of HIV strains have been 
described. The deduced protein sequence for TAT we have used in the design 
of a synthetic TAT gene was taken from the first published sequence 
(Ratner et al. Nature 313, 277-284 (1985)). 
The function of TAT as an anti-terminator has been established more 
recently (Wright et al. Science 234, 988-992 (1986), Kao et al. Nature 
330, 489-493 (1987)). 
The construction of a synthetic gene encoding TAT is not described in any 
of the above documents. 
In order to facilitate the the further delineation of the mode of 
trans-activation by TAT, the use of TAT and TAR in the construction of 
inducible expression systems and the production of TAT protein for 
structural and immunological studies an improved novel synthetic modular 
gene for TAT is sought. 
It is by no means easy to predict the design of an improved gene for TAT, 
since the factors that determine the expressibility of a given DNA 
sequence are still poorly understood. Furthermore, the utility of the gene 
in various applications will be influenced by such considerations as codon 
usage and restriction sites. The present invention relates to synthetic 
TAT genes which are distinct from the natural TAT gene and have advantages 
in the ease with which they can be modified due to the presence of useful 
restriction sites. 
According to a second aspect of the invention, there is provided DNA coding 
for TAT and having restriction sites for the following enzymes: 
##STR1## 
According to a third aspect of the invention, there is provided DNA 
including the following sequence: 
##STR2## 
The design for the synthetic tat genes was based on the amino acid 
sequence deduced from the published cDNA sequence (see FIG. 1). It was 
found possible to assign appropriate codons to the synthetic gene aiming 
for a compromise between E. coli and yeast codon bias. For the codons 
where no compromise choice was available a strategy of alternating the 
codon choice was devised such that runs of sub-optimal codons for one 
organism were avoided. In addition a number of restriction sites were 
built into the sequence to facilitate subsequent manipulation of segments 
of the tat gene. For the preferred sequence, the codon selection was then 
randomised within this set of constraints by computer and the sequence 
checked finally to ensure that there were no regions of extensive direct 
or inverted repeats. To simplify the incoporation of the tat gene into 
expression vectors a number of flanking restriction sites were chosen 
including an upstream HinDIII site and downstream BamHI and EcoRI sites. 
Provision was also made for the construction of tat fusion derivatives 
with or without the initiator methionine through the inclusion of NcoI and 
BsoMI sites that encompass the initiator ATG. These sites allow the 
retention of a reasonable Kozak sequence that may be important for 
applications involving the expression of the synthetic gene in mammalian 
cells. 
Synthetic genes in accordance with the invention are designed primarily for 
expression in yeast and E. coli but we would expect them to be capable of 
expression in other systems including mammalian and insect cells. 
According to a fourth aspect of the invention, there is provided a genetic 
construct comprising DNA according to the first or second aspect or a 
fragment thereof. The fragment may comprise at least 10, 20, 30, 40 or 50 
nucleotides. A genetic construct in accordance with the third aspect may 
be a vector, such as a plasmid, cosmid or phage. 
According to a fifth aspect of the invention, there is provided a process 
for the preparation of DNA in accordance with the second or third aspect 
or a genetic construct in accordance with the fourth aspect, the process 
comprising coupling successive nucleotides and/or ligating appropriate 
oligomers. It is preferred, however, that a method in accordance with the 
first aspect be used. 
The invention also relates to other nucleic acid (including RNA) either 
corresponding to or complementary to DNA in accordance with the second or 
third aspects.

CONSTRUCTION OF THE GENE 
The top strand of the desired gene sequence was divided into 2 large 
oligodeoxyribonucleotides (oligomers) BB512 and BB513 as depicted in FIG. 
3. The ligation of these two large oligomers and their subsequent cloning 
into the plasmid vector pUC18 was then accomplished through the use of 
small complementary bridging oligomers. BB511 is a 16mer that is 
complementary to the 3' 8 bases of BB512 and the 5' 8 bases of BB513. 
BB514 is an 11mer that serves as an adapter by annealing to the 5'end of 
BB512 in such a way as to leave a four base HindIII compatible cohesive 
end. Similarly, BB515 is a 14mer that anneals to the 3' end of BB513 to 
provide an EcoRI cohesive end. 
The oligomers were synthesised by automated solid phase phophoramidite 
chemistry. Following de-blocking and removal from the controlled pore 
glass support the oligomers were purified on denaturing polyacrylamide 
gels, further purified by ethanol precipitation and finally dissolved in 
water prior to estimation of their concentration. 
To minimise the possibility of mis-ligation only BB513, the 3' of the two 
large oligomers, was kinased. All five oligomers were annealed with the 
three small adapter and bridging oligomers in two fold molar excess. The 
annealed mixture was then ligated directly to EcoRI/HinDIII cut DNA of the 
plasmid vector pUC18. This procedure allows the sense strand of tat to be 
ligated into the vector resulting in plasmid DNA carrying a single 
stranded gap covering the tat gene. This procedure relies on in vivo gap 
repair to fill in the single stranded region. The ligated product was 
transformed into HW87 and plated on L-agar plates containing 100 
mcg.ml.sup.-1 ampicillin. Colonies containing potential clones were then 
grown up in L-broth containing ampicillin at 100 mcg.ml.sup.-1 and plasmid 
DNA isolated. Positive clones were identified by direct dideoxy sequence 
analysis of the plasmid DNA using the 17 base universal primer, a reverse 
sequencing primer complementary to pUC18 on the other side of the 
polylinker region. Some of the oligomers employed in the assembly of the 
gene were also used as internal sequencing primers. One tat clone was 
subsequently re-sequenced on both strands to confirm that no mutations 
were present. 
METHODS 
All the basic techniques of genetic manipulation used in the manufacture of 
this gene are well known to those skilled in the art of genetic 
engineering. A description of most of the techniques can be found in one 
of the following laboratory manuals: Molecular Cloning by T. Maniatis, E. 
F. Fritsch and J. Sambrook published by Cold Spring Harbor Laboratory, Box 
100, New York, or Basic Methods in Molecular Biology by L. G. Davis, M. D. 
Dibner and J. F. Battey published by Elsevier Science Publishing Co. Inc. 
New York. 
Additional and modified methodologies are detailed below. 
1) Oligonucleotide Synthesis 
The oligonucleotides were synthesised by automated phosphoramidite 
chemistry using cyanoethyl phosphoramidtes. The methodology is now widely 
used and has been described (Beaucage, S. L. and Caruthers, M. H. 
Tetrahedron Letters 24, 245 (1981)). 
2) Purification of Oligonucleotides 
The oligonucleotides were de-protected and removed from the CPG support by 
incubation in concentrated NH.sub.3. Typically, 50 mg of CPG carrying 1 
micromole of oligonucleotide was de-protected by incubation for 5 hr at 
70.degree. in 600 mcl of concentrated NH.sub.3. The supernatant was 
transferred to a fresh tube and the oligomer precipitated with 3 volumes 
of ethanol. Following centrifugation the pellet was dried and resuspended 
in 1 ml of water. The concentration of crude oligomer was then determined 
by measuring the absorbance at 260 nm. 
For gel purification 10 absorbance units of the crude oligonucleotide were 
dried down and resuspended in 15 mcl of marker dye (90% de-ionised 
formamide, 10 mM tris, 10 mM borate, 1 mM EDTA, 0.1% bromophenol blue). 
The samples were heated at 90.degree. for 1 minute and then loaded onto a 
1.2 mm thick denaturing polyacrylamide gel with 1.6 mm wide slots. The gel 
was prepared from a stock of 15% acrylamide, 0.6% bisacrylamide and 7M 
urea in 1.times. TBE and was polymerised with 0.1% ammonium persulphate 
and 0.025% TEMED. The gel was pre-run for 1 hr. The samples were run at 
1500 V for 4-5 hr. The bands were visualised by UV shadowing and those 
corresponding to the full length product cut out and transferred to 
micro-testubes. The oligomers were eluted from the gel slice by soaking in 
AGEB (0.5 M ammonium acetate, 0.01 M magnesium acetate and 0.1% SDS) 
overnight. The AGEB buffer was then transferred to fresh tubes and the 
oligomer precipitated with three volumes of ethanol at -70.degree. for 15 
min. The precipitate was collected by centrifugation in an Eppendorf 
microfuge for 10 min, the pellet washed in 80% ethanol, the purified 
oligomer dried, redissolved in 1 ml of water and finally filtered through 
a 0.45 micron micro-filter. The concentration of purified product was 
measured by determining its absorbance at 260 nm. 
3) Kinasing of Oligomers 
250 pmole of BB513 was dried down and resuspended in 20 mcl kinase buffer 
(70 mM Tris pH 7.6, 10 mM MgCl.sub.2, 1 mM ATP, 0.2 mM spermidine, 0.5 mM 
dithiothreitol). 10 u of T4 polynucleotide kinase was added and the 
mixture incubated at 37.degree. for 30 min. The kinase was then 
inactivated by heating at 85.degree. for 15 min. 
4) Annealing 
10 pmol of BB512 and BB513 were mixed with 20 pmol each of oligomers BB511, 
BB514 and BB515. The mixture was heated to 90.degree. for 10 minutes and 
then cooled slowly to room temperature to allow the oligomers to anneal. 
5) Ligation 
The annealed oligomers were then mixed with 10 X ligase buffer to give a 
final ligase reaction mixture (50 mM Tris pH 7.5, 10 mM MgCl.sub.2, 20 mM 
dithiothreitol, 1 mM ATP. T4 DNA ligase was added at a rate of 100 u per 
50 mcl reaction and ligation carried out at 15.degree. for 4 hr. 
6) Cloning of Fragment 
0.5 mcg of pUC18 DNA was prepared by cleavage with HinDIII and BamHI as 
advised by the suppliers. The digested DNA was run on an 0.8% LGT gel and 
the vector band purified as described below. 
20 ng of cut vector DNA was then ligated to various quantities of annealed 
tat DNA ranging from 2 to 20 ng for 4 hr using the ligation buffer 
described above. The ligation products were used to transform competent 
HW87 as has been described. Ampicillin resistant transformants were 
selected on L-agar plates containing 100 mcg.ml.sup.-1 ampicillin. 
7) Agarose Gel Electrophoresis 
The digested vector DNA was purified on a 0.8% low gelling temperature 
agarose gel in 1.times. TBE buffer (0.094 M Tris pH8.3, 0.089 M boric 
acid, 0.25 mM EDTA) containing 0.5 mcg.ml.sup.-1 ethidium bromide. The 
band corresponding to linearised plasmid DNA was excised and the DNA 
extracted as below. 
8) Isolation of Vector DNA 
The volume of the gel slice was estimated from its weight and then melted 
by incubation at 65.degree. for 10 min. The volume of the slice was then 
made up to 400 ul with TE (10 mM Tris pH 8.0, 1 mM EDTA) and Na acetate 
added to a final concentration of 0.3 M. 10 mcg of yeast tRNA was also 
added as a carrier. The DNA was then subjected to three rounds of 
extraction with equal volumes of TE equilibrated phenol followed by three 
extractions with ether that had been saturated with water. The DNA was 
precipitated with 2 volumes of ethanol, centrifuged for 10 min in a 
microfuge, the pellet washed in 70% ethanol and finally dried down. The 
DNA was taken up in 20 mcl of TE and 2 mcl run on a 1% agarose gel to 
estimate the recovery of DNA. 
9) Isolation of Plasmid DNA 
Plasmid DNA was prepared from the colonies containing potential tat clones 
essentially as described (Ish-Horowicz, D., Burke, J. F. Nucleic Acids 
Research 9 2989-2998 (1981). 
10) Dideoxy Sequencing 
The protocol used was essentially as has been described (Biggin, M. D., 
Gibson, T. J., Hong, G. F. P.N.A.S. 80 3963-3965 (1983)). The method was 
modified to allow sequencing on plasmid DNA as described (Guo, L-H., Wu, 
R. Nucleic Acids Research 11 5521-5540 (1983). 
11) Transformation 
Transformation was accomplished using standard procedures. The strain used 
as a recipient in the cloning was HW87 which has the following genotype: 
##STR3## 
Any other standard cloning recipient such as HB101 would be adequate.