Cloning of random-sequence oligodeoxynucleotides

Methods are described for cloning random or highly degenerate nucleotide (nt) sequences. The procedures use synthetically derived mixtures of oligodeoxynucleotides (oligos) whose heterogeneous central portions are bounded at their 5' and 3' ends by sequences recognized by restriction endonucleases. Oligo collections of defined length and nt composition are synthesized by utilizing appropriate concentrations of all four nucleotide precursors during each addition step for the central region. Single-stranded oligos with appropriate 5' and 3' ends can be ligated directly, although inefficiently, into double-stranded (ds) DNA molecules with complementary 5' and 3' extensions produced by restriction endonuclease cleavage. A more general and efficient method is to convert the oligo into a ds form by incubating it with the Klenow (large) fragment of Escherichia coli DNA polymerase I. If the 3' ends are palindromic, two oligo molecules will serve as mutual primers for polymerization. The resulting products are ds molecules containing two oligo units separated by the original 3' restriction site and bounded at each end by the original 5' restriction site. After appropriate restriction endonuclease cleavage, oligo units can be cloned by standard procedures. Analysis of 26 recombinant M13 phages indicates that the nt sequences of the cloned oligos are in good accord with what was expected on a random basis.     

An MboII/FokI trimming plasmid allowing consecutive cycles of precise 1- to 12-base-pair deletions in cloned DNA

A novel trimming plasmid has been designed which allows, in a preprogrammed fashion, the precise deletion of up to 12 bp per cleavage cycle, from one end of a cloned fragment. The plasmid, which carries the dhfr gene, contains unique recognition sites for two class-IIS restriction enzymes, MboII and FokI, which are arranged in the form of a cassette, so that consecutive cleavages with these endonucleases, followed by blunting with mung bean nuclease (MB), will precisely delete 12 bp of adjacent cloned DNA. When either MboII or FokI is used alone (followed by MB), 1 or 4 bp are removed, respectively. The final step in the trimming cycle is religation of the plasmid with T4 ligase. After required number of cycles, plasmids were transformed into Escherichia coli C600, and transformants selected by resistance to trimethoprim. Since the MboII/FokI cassette remains intact during these operations, one can repeat the cycle, consisting of cleaving, MB blunting and religation, several times, each time removing up to 12 bp from the cloned target DNA. Examples are provided of one-, two- and three-cycle trimmings.     

Plasmids pMB123 and pMB124--vectors for obtaining subfragments of DNA with random "sticky" ends

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.     

A novel multistep method for generating precise unidirectional deletions using BspMI, a class-IIS restriction enzyme

A novel approach is described that permits the introduction of unidirectional deletions into a cloned DNA fragment, in a precisely controlled manner. The method is based on the use of a special vector and a class-IIS restriction endonuclease, BspMI, which produces staggered cuts 4 and 8 nucleotides (nt) to the 3' from its recognition site 5'-ACCTGC-3'. The DNA fragment is inserted into the pUC19-based plasmid, which contains a unique BspMI recognition site, and the appropriate number of cleavage-and-deletion cycles is performed, each cycle removing 4 bp. Since the recognition site is not affected by the BspMI cleavage, no recloning of the DNA fragment is necessary. Each cycle consists of BspMI cleavage, removal of the 4-nt single-stranded cohesive ends with mung bean nuclease (MB), and blunt-end ligation to recircularize the plasmid. The shortened plasmid is reintroduced into the host, after one or after several such 4-bp deletion cycles. When DNA is inserted into the multiple cloning site in the lacZ alpha gene, the progress of 4-bp removal can be followed by determining the Lac phenotype, since removal of multiples of 3 bp retains the reading frame while other kinds of deletions distort (or restore) the reading frame. Loss of pre-existing restriction sites or creation of new ones also permits monitoring the progress of the deletion process.(ABSTRACT TRUNCATED AT 250 WORDS)     

A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2.

In this paper we describe the synthesis and cloning of a short segment of DNA complementary to the region immediately adjacent to the EcoRI insertion site in the single-stranded bacteriophage vector M13mp2. This segment is useful as a "universal" primer for DNA sequencing by the dideoxynucleotide chain termination method; the template can be any DNA species cloned in M13mp2 or its derivatives. The primer has been cloned into the tetracycline resistance gene of plasmid pBR322 as one strand of a 26 bp EcoRI/BamHI fragment. This fragment may be readily prepared from an EcoRI + BamHI restriction digest of the parent plasmid (designated pSP14) by a simple size fractionation.     

An alternative approach in gene synthesis: use of long selfpriming oligodeoxynucleotides for the construction of double-stranded DNA

A novel approach for the synthesis of double-stranded DNA fragments from only one long oligodeoxynucleotide (oligo) is presented. The basic strategy is to use oligos which possess a short inverted repeat at their 3' end resulting in the formation of a hairpin structure. The 3' end of this hairpin then serves as a primer in the Klenow (large) fragment of E. coli DNA polymerase I-mediated synthesis of the second DNA strand. Removal of the loop structure as well as generation of sticky ends for subsequent cloning is achieved by digestion with restriction enzymes. Several oligos ranging in size from 130 to 147 nt were synthesized and successfully used in the cloning of gene fragments of up to 120 bp in length. Furthermore, a strategy for the simultaneous cloning of two synthetic DNA fragments is outlined yielding even larger gene fragments. By sequential cloning of these gene fragments the methodology presented here will allow the synthesis of genes of any size. The proposed methodology should also be useful for site-directed mutagenesis as well as saturation mutagenesis.     

Resolution of sequencing ambiguities: a universal FokI adapter permits Maxam-Gilbert re-sequencing of single-stranded phagemid DNA

We propose a method to resolve ambiguities encountered when single-stranded (ss) phagemid DNA templates are sequenced by the dideoxy method. A single oligodeoxyribonucleotide (oligo) is synthesized with the following features: (i) the 20 nucleotides (nt) at the 5'-end form a double-stranded hairpin containing a FokI restriction site, exactly as previously described by Podhajska and Szybalski [Gene 40 (1985) 175-182]; (ii) the 23 nt at the 3'-end hybridize to the (+)strand of ss phagemid DNA in the region complementary to the M13 universal sequencing primer. In a simple one-tube set of reactions, ss phagemid DNA is annealed to this oligo, cleaved by FokI at a unique site outside the vector multiple cloning site and then labelled at this unique site by Klenow polymerase and [alpha-32P]dCTP. These reactions provide a convenient route by which Maxam-Gilbert chemical degradation sequencing methods can be used to resolve ambiguities encountered in the dideoxy-sequencing of a unidirectional deletion series already prepared in popular phagemid vectors. A single oligo allows labelling of all members of a deletion series. A second universal oligo allows the same set of reactions to be applied to inserts cloned into (-)strand phagemids.     

A method for the generation of small pre-determined deletions in plasmid DNA: deletion analysis of the tetR region of vector pBR322

A general method is described that allows precise deletion of a chosen restriction fragment(s) from a plasmid having many cleavage sites for that restriction enzyme. The DNA to be deleted is first separated from the rest of the plasmid on a larger DNA fragment contained between two different unique restriction sites. This fragment is then subdigested by the restriction endonuclease of interest, which recognises two or more tetranucleotide (cohesive end or blunt end) sequences on the fragment, and is recloned between the two original unique restriction sites. The method is rapid, efficient, and the results are predictable. Examples are given in which predetermined HpaII (9 bp, 147 bp), TaqI (141 bp) and AluI (15 bp, 403 bp) fragments have been selectively removed from the tetR region of plasmid pBR322.     

Cloning a defined region of DNA using a limited action of DNA polymerase: application to dissection of hepatitis B virus surface antigen gene

Using dodecadeoxynucleotides as primers for DNA synthesis and 3'-o-chlorophenyl-phosphorylated dodecadeoxynucleotides as "stoppers" for chain elongation, pre-defined regions of a gene previously cloned in M13 single-stranded (ss) DNA phage were converted into double-stranded (ds) DNA utilizing the action of the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). The resulting ds DNA was freed from the ss region by S1 nuclease treatment. This method can be used to obtain DNA fragments of any size with pre-defined 5' and 3' ends. About 15% of the input ss DNA template molecules are converted into ds DNA fragments. This technique was used to synthesize several DNA fragments from different portions of the hepatitis B virus surface antigen (HBsAg) gene. The products were then ligated into a yeast plasmid vector that carries the E. coli lacZ gene which is located downstream from the yeast acid-phosphatase promotor. Using this system, several fragments of HBsAg were produced in the form of beta-galactosidase fused protein.     

A method for cloning mixtures of long,synthetic oligodeoxynucleotides

A method is described for cloning synthetic oligodeoxynucleotides, which can theoretically be of any length. The method requires only a single oligodeoxynucleotide strand and a vector with two unique restriction sites, one of which is for an enzyme that generates 3′ protruding ends. A mixture of unpurified oligonucleotides containing a wild-type genetic regulatory sequence of the Escherichia coli gnd gene and two mutations of it was cloned into a plasmid carrying a gnd-lacZ protein fusion. Individual cloned oligonucleotides were readily identified by direct DNA sequencing of plasmid templates. The method is rapid, efficient, and has application to gene synthesis and site-directed mutagenesis.     

NtSKP1基因的反义载体构建及转基因烟草的产生

根据枯斑三生烟SKP1基因(NtSKP1)的序列,设计一对分别含有特定酶切位点的特异引物,以重组质粒pMD18-SKP1为模板,扩增目的基因(约473bp)片段。将反向目的片段插入中间载体pHANNIBAL的内含子右侧,再经NotⅠ酶切回收约3443bp的目的片段,插入到双元载体质粒pART27中,成功构建了含NtSKP1基因片段反向序列的植物表达载体pART27-skp1a,其转录产物能减弱目的基因的表达。将pART27-skp1a质粒导人根癌农杆菌LBA4404中并转化烟草叶片细胞,经选择分化培养,获得转基因烟草。     

嗜麦芽假单胞菌酪氨酸酶基因在大肠杆菌中的克隆与表达

酪氨酸酶基因（ｍｅｌ）编码的酪氨酸酶是合成黑色素的关键酶。用鸟枪法分离嗜麦芽假单胞菌的ｍｅｌ基因：以ｐＵＣ１８为载体,Ｅ．ｃｏｌｉ ＨＢ１０１为受体菌,在加有一定量的Ａｍｐ和Ｌ－ｔｙｒ的酪素平板上筛选到分泌可溶性黑色素的转化子,所含重组质粒ｐＷＳＹ约７００ｂｐ的外源ＤＮＡ片段上携有ｍｅｌ基因,该片段无ＢａｍＨＩ、ＨｉｎｄＩＩＩ、ＥｃｏＲＩ、ＢｃｌＩ等酶的识别位点。Ｓｏｕｔｈｅｒｎ杂交证实此片段确实     

Effective synthesis and cloning of the human interleukin-2 gene and its analog: expression of the interleukin-2 gene in E. coli cells]

Artificial DNA fragments encoding human interleukin-2 (133 a.a.) and its analogue (deletion of 14 C-terminal a.a.) were prepared by means of the DNA polymerase I mediated extension of synthetic polynucleotides having short overlapping sequences at their 3'-ends. The fragments were cloned in specially designed pFH-type plasmids and then excised by the FokI and other restriction endonucleases to yield the subfragments with the structurally predetermined 5'-unique cohesive ends. The complete synthetic gene was constructed by one or two-step ligation. The expressed IL-2 was tested by analysing the T-cell proliferation activity of E.coli crude lisates containing the pEXIL2 expression plasmid.     

HSV-tk基因逆转录病毒重组体的构建与DNA序列分析

目的　构建含有单纯疱疹病毒Ⅰ型胸苷激酶 (HSV1 tk)基因的逆转录病毒重组载体pLXSN TK。方法设计一对寡核苷酸引物 ,用PCR方法从质粒pHSV10 6中特异扩增HSV tk基因片段 ( 1168bp) ,分别用BamHI和Eco RI酶切后 ,定向连接到质粒pLXSN中 ,转化宿主菌TG1,分别用上述内切酶 ,PCR和DNA测序鉴定重组质粒。结果　酶切鉴定所切下的片段和PCR扩增的片段大小均与预计相符 ,测序结果与文献报道序列及预计结果一致 ,证实符合表达框架。结论　成功构建了HSV tk嵌合重组质粒pLXSN TK。     

Total DNA synthesis and cloning in Escherichia coli of a gene coding for the human growth hormone releasing factor

A DNA containing a sequence coding for the human growth hormone releasing factor (hGRF) has been obtained by enzymatic assembly of chemically synthesized DNA fragments. The synthetic gene consists of a 140 base-pair fragment containing initiation and termination signals for translation and appropriate protruding ends for cloning into a newly constructed plasmid vector (pULB1219). Eleven oligodeoxyribonucleotides, from 14 to 31 bases in length, sharing pairwise stretches of complementary regions of at least 13 bases were prepared by phosphotriester solid-phase synthesis. The DNA sequence was designed to take into account the optimal use of E. coli codons. Oligomers were annealed in one step and assembled by ligation. The DNA fragment of the expected size (140 bp) was recovered and cloned into the pULB1219 vector. The expected sequence was confirmed by DNA sequencing.     

麦迪霉素4"-O-丙酰基转移酶(mpt)基因结构的研究

对含有麦迪霉素4"-O-丙酰基转移酶(mpt)基因的BamHI-BamHI 8.0kb的DNA片段进行限制性酶切分析,绘制出了含有21个酶切位点的限制性酶切图谱。以含有碳霉素异戊酰基转移酶基因(CarE)的2.4kb DNA片段为探针,经Southern blot分子杂交,将mpt定位于一个EcoRI-EcoRI-Pstl 3.0kb的DNA片段上,将该片段克隆至大肠杆菌/链霉菌穿梭质粒载体pWHM3上,获得重组质粒pWFPE。含有pWFPE的螺旋霉素产生菌产二素链霉菌(S.ambofaciens)及变铅青链霉菌(J.lividans)TK24均可将内源产生的或外源加入的螺旋霉素酰化为4"-O-丙酰螺旋霉素。对EcoRI-EcoRI-PstI 3.0kbDNA片段上mpt基因进行序列分析,在该片段上有一个开放阅读框架,它以ATG为起始密码子,以TGA为终止密码子,与其序列对应的编码产物含有388个氨基酸。Mpt基因的G+C mol％为68.0,密码子第三位上G+C mol％为91.5。Mpt基因编码的氨基酸序列与CarE基因编码的氨基酸序列的相同性为67.6％,相似性为86.4％。在起始密码子上游6bp处存在…     

A simple and efficient enzymatic method for covalent attachment of DNA to cellulose. Application for hybridization-restriction analysis and for in vitro synthesis of DNA probes.

Single-stranded DNAs (ssDNAs) were covalently bound by a simple and efficient enzymatic method to a solid support matrix and used to develop several new procedures for gene analysis. The novel procedure to prepare a ssDNA stably coupled to a solid support employed T4 DNA ligase to link covalently oligo (dT)-cellulose and (dA)-tailed DNA. Beginning with essentially any double stranded DNA the procedure generates a ssDNA linked by its 5' end to a cellulose matrix in a concentration of over 500 ng per mg. DNA from the plasmid pBR322 (4300 bp) and a fragment of the beta-globin gene (1800 bp) were coupled to the solid support and used for several experiments. The ssDNAs on the cellulose efficiently hybridized with as little as 5 pg of complementary double-stranded DNAs. The DNA hybrids formed on the solid support were specifically and efficiently cleaved by restriction endonucleases. These specific restriction cuts were utilized for the diagnosis of correct sequences. In addition, the ssDNA on the solid support served as an efficient template for the synthesis of complementary ssDNAs. The complementary synthesized ssDNAs were uniformly labeled, more than two kilobases in size, and largely full length. About 85% of the ssDNA linked to cellulose was available for the synthesis of complementary DNA, and after strand-separation, the preparation was reusable for the synthesis of additional complementary DNA.     

Synthesis of DNA coding for human proinsulin

A chemical-enzymatic synthesis of 271- and 286-bp DNA duplexes, each of which contains the entire sequence coding for human proinsulin has been accomplished. In addition to the coding sequence, the 271-bp fragment carries translation initiation and termination signals plus EcoRI-HindIII restriction enzyme sites for insertion into an appropriate plasmid vector. The 286-bp fragment also contains a Shine-Dalgarno (SD) sequence preceding an ATG codon. Employing the 286-bp polynucleotide, the 568-bp tandem proinsulin gene has been obtained. The synthesis of these DNA fragments involved preparation of 42 oligonucleotides by a rapid N-methylimidazolide phosphotriester method and enzymatic conversion of the oligonucleotides into the gene subfragments, which were cloned separately and fused to yield the desired DNAs coding for proinsulin. The proinsulin gene fragments were cloned in Escherichia coli and shown to have the correct sequences.