Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors
AssigneeMedical Research Council
InventorsGregory Paul Winter, Elizabeth Sally Ward, Detlef Gussow
Life of this patent
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Abstract
The present invention relates to single domain ligands derived from molecules in the immunoglobulin (Ig) superfamily, receptors comprising at least one such ligand, methods for cloning, amplifying and expressing DNA sequences encoding such ligands, preferably using the polymerase chain reaction, methods for the use of said DNA sequences in the production of Ig-type molecules and said ligands or receptors, and the use of said ligands or receptors in therapy, diagnosis or catalysis.
Claims32
A single domain ligand consisting of at least part of the variable domain of one chain of a molecule from the immunoglobulin (Ig) superfamily.
A method of cloning a sequence (the target sequence) which encodes at least part of the variable domain of an Ig superfamily molecule, which method comprises:
- (a) providing a sample of double stranded (ds) nucleic acid which contains the target sequence;
- (b) denaturing the sample so as to separate the two strands;
- (c) annealing to the sample a forward and a back oligonucleotide primer, the forward primer being specific for a sequence at or adjacent the 3′ end of the sense strand of the target sequence, the back primer being specific for a sequence at or adjacent the 3′ end of the antisense strand of the target sequence, under conditions which allow the primers to hybridise to the nucleic acid at or adjacent the target sequence;
- (d) treating the annealed sample with a DNA polymerase enzyme in the presence of deoxynucleoside triphosphates under conditions which cause primer extension to take place;
- and
- (e) denaturing the sample under conditions such that the extended primers become separated from the target sequence.
A method of cloning a sequence (the target sequence) which encodes at least part of the variable domain of an Ig superfamily molecule, which method comprises:
- (a) providing a sample of double stranded (ds) nucleic acid which contains the target sequence;
- (b) denaturing the sample so as to separate the two strands;
- (c) annealing to the sample a forward and a back oligonucleotide primer, the forward primer being specific for a sequence at or adjacent the 3′ end of the sense strand of the target sequence, the back primer being specific for a sequence at or adjacent the 3′ end of the antisense strand of the target sequence, under conditions which allow the primers to hybridise to the nucleic acid at or adjacent the target sequence;
- (d) treating the annealed sample with a DNA polymerase enzyme in the presence of deoxynucleoside triphosphates under conditions which cause primer extension to take place;
- (g) treating the sample of ds cDNA with traces of DNAse in the presence of DNA polymerase I to allow nick translation of the DNA;
- and
- (h) cloning the ds cDNA into a vector.
A species specific general oligonucleotide primer or mixture of such primers useful for cloning at least part of a variable domain encoding sequence from an animal of that species.
Dependencies are inferred from the claim text.