Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1994-11-14
1997-02-18
Fleisher, Mindy
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
4351721, 435183, 536 242, 935 23, C12N 1500, C07H 2104
Patent
active
056041225
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an improved method of cloning DNA into a vector, and to the products enabling the method to be conducted.
Any DNA cloning procedure has four essential parts: a method for generating DNA fragments; reactions which join foreign DNA to the vector; a means of introducing the artificial recombinant into a host in which it can replicate, and a method of selecting or screening for a clone of recipient cells that has acquired the recombinant.
This invention is concerned in particular with reactions which join foreign DNA (DNA to be cloned) to the vector. Consequently the choice of restriction endonucleases, the use of specific linkers or adaptor molecules and the choice of specific vectors is crucial.
The method, which can use novel linkers and vectors, can be applied to cloning cDNA, genomic DNA cloning, or the products of PCR reactions.
There are currently two methods of joining DNA in vitro, thereby enabling cloning; these suffer a number of drawbacks.
The first of these methods, and that used with genomic DNA and cDNA, utilizes the ability of DNA ligase to covalently join the cohesive ends produced by the action of certain restriction enzymes on DNA. With cohesive ends one strand of the DNA is longer than the other and therefore protrudes and forms an overhang of DNA.
The second, also used with genomic DNA and cDNA, utilizes the ability of DNA ligase to catalyse the formation of phophodiester bonds between DNA blunt ends.
The reaction that DNA ligase carries out on blunt or cohesive ended DNA is substantially the same, the only difference being that in the case of cohesive ends the ends to be joined are held in place prior to ligation by their inherent cohesiveness whereas this is not the case when blunt ends are to be joined.
DNA ligase is most commonly used to covalently bond a restriction endonuclease cut DNA fragment to a vector cut with the same restriction endonuclease. The problem with this method is that recircularization of both linearised vector and DNA fragments can occur.
Since the ligation reaction is often performed in dilute solutions the circularization of linear fragments is relatively favoured due to the reduced frequency of intermolecular annealing. This in turn affects the number of recombinants. The recircularization of the vector can be avoided by treating the linearised vector with alkaline phosphatase to remove the 5' terminal phosphate groups thus preventing self ligation.
A modification of the procedure for use with cDNA uses DNA ligase to add linkers to the flush ends of eDNA. Linkers are short pieces of double stranded DNA which contain one or more restriction sites for endonuclease restriction enzymes. These are ligated on to the cDNA (a reaction which proceeds at an acceptable rate because of the high concentration of linker ends compared to that of the cDNA ends). The DNA is then restricted with the corresponding enzyme which products a cohesive end on the DNA. DNA in this form can then be ligated into a vector because the cohesive ends of the foreign DNA make the ligation proceed at an acceptable rate despite the relatively low concentration of both vector and foreign DNA ends.
To prevent the enzyme used to cleave the linkers from restricting the foreign DNA prior to the addition of the linkers the foreign DNA is "methylated", this involves enzymically adding methyl groups on certain nucleotides within the enzymes recognition sequences. This will prevent the foreign DNA (cDNA) from being restricted despite it containing the recognition sequence of the enzyme. Thus when the linkers are restricted to yield a cohesive end the enzyme recognition sites within the foreign DNA are protected from restriction.
The difficulties associated with the aforementioned strategy involving linkers is that the linkers can ligate together to produce a "pseudo insert" a piece of DNA which is made up of a linker concatemer and does not contain any foreign DNA at all. Also, the ends of one piece of foreign DNA can ligate to the end of another and thus multiple inserts a
REFERENCES:
Lathe et al. Linker Tailing: Unphosphorylated linker oligonucleotides for joining DNA termini DNA vol. 3 173-182 1984.
Lathe et al. DNA enginneering: the use of enzymes, chemicals and oligonucleotides to restructure DNA sequences in vitro in: Genetic Engineering 4 Robert Williamson Ed. Academic Press London 2-56 1983.
Maniatis et al. Molecular Cloning a Laboratory Manual Cold Spring Harbor Laboratory 17-44 1982.
Life Technologies Catalog pp. 6-15, 6-17, 6-19, 6-34, and 6-25 1993.
Calleja et al. A new endonuclease recognizing the deoxynucleotide sequence CCNNGG from the cyanobacterium Synechocystis 6701 Nucleic Acids Res. vol. 13 6745-6751 1985.
Razin et al. Studies on the biological role of DNA methylation; IV. Mode of methylation of DNA in E. coli cells vol. 8 1783-1792 1980.
Dahl et al., "The Use of Genomic Libraries for the Isolation and Study of Eucaryotic Genes," Genetic Engineering, 2 (Williamson, ed.), Academic Press Inc., London, U.K., 1981, pp. 70-73.
Seth, "A New Method for Linker Ligation," Chemical Abstracts, 102 (19) Abstract 161508m, 163 (1985).
New England Biolabs Inc. Product Catalog, Beverly, MA (1990-1991), pp. 20, 42-45, 81.
Brusca John S.
Fleisher Mindy
The University of Hull
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