US4888286A
Expired / Lapsed
US
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Production of gene and protein analogs through synthetic gene design using double stranded synthetic oligonucleotides
Assignee: Creative Biomolecules Inc
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Filing: 1987-03-24
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Granted: 1989-12-19
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Expires: 2007-03-24
Abstract
Abstract Methods and compositions are provided for the production of gene mutations and protein analogs using synthetic gene design and double-stranded synthetic oligonucleotide cassettes. The disclosure provides synthetic structural genes, synthetic double-stranded oligonucleotide cassettes and methods for the creation of synthetic gene analogs and polypeptide analogs by shortening or altering the native amino acid sequence.
Inventors
- 👤 Roberto Crea
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 01:45
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/102; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/60; C07K14/65
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
156 words
A method of altering the nucleotide sequence of native double-stranded DNA encoding a protein, to prepare synthetic double-stranded DNA capable of expressing an analog of said protein, said method comprising the steps of: providing a synthetic double-standard DNA comprising a sequence of codons capable of expressing the amino acids of said protein and providing a plurality of non native restriction endonuclease sites; digesting said synthetic double stranded DNA with a first restriction endonuclease having a first cleavage recognition site within said double stranded DNA; digesting said synthetic double-stranded DNA with a second restriction endonuclease having a second cleavage recognition site proximate to said first site, whereby at least major and minor structural gene fragments are created; and ligating to said major genre fragment a synthetic double-stranded oligonucleotide having termini complementary to said first and second cleavage sites, said synthetic double-stranded oligonucleotide having a nucleotide sequence distinct from the nucleotide sequence of said minor structural gene fragment.
Claim 2
Independent Claim
156 words
A method of altering the nucleotide sequence of native double-stranded DNA encoding a protein, to prepare synthetic double-stranded DNA capable of expressing an analog of said protein, said method comprising the steps of: providing a synthetic double-standard DNA comprising a sequence of codons capable of expressing the amino acids of said protein and providing a plurality of non native restriction endonuclease sites; digesting said synthetic double stranded DNA with a first restriction endonuclease having a first cleavage recognition site within said double stranded DNA; digesting said synthetic double-stranded DNA with a second restriction endonuclease having a second cleavage recognition site proximate to said first site, whereby at least major and minor structural gene fragments are created; and ligating to said major genre fragment a synthetic double-stranded oligonucleotide having termini complementary to said first and second cleavage sites, said synthetic double-stranded oligonucleotide having a nucleotide sequence distinct from the nucleotide sequence of said minor structural gene fragment.
Claim 3
Dependent Claim
41 words
A method as recited in claim 1 wherein the major gene fragment comprises a plurality of fragments generated in said digestion steps, said method comprising the additional step of reassembling said plurality of fragments upon ligation with said synthetic double-stranded oligonucleotide.
Claim 4
Dependent Claim
45 words
A method as recited in claim 1 wherein the nucleotide sequence of said synthetic DNA defines a first restriction site specific to said first restriction endonuclease, and a second restriction site specific to said second restriction endonuclease which is distinct from said first restriction site.
Claim 5
Dependent Claim
25 words
A method as recited in claim 1 wherein said synthetic double-stranded DNA codes for the amino acid sequence identified as human pancreatic Growth Hormone-Releasing Factor.
Claim 6
Dependent Claim
28 words
A method as recited in claim 4 wherein said double-stranded oligonucleotide comprises a sequence designed to replace the internal methionine in said human pancreatic Growth Hormone-Releasing Factor polypeptide.
Claim 7
Dependent Claim
77 words
A method as recited in claim 5 wherein said double-stranded oligonucleotide comprises: ______________________________________
5'- G G A T A T C X Y Z T - 3'
A C G T C C T A T A G X' Y' Z' A G A T C - 5'
______________________________________ wherein X, Y and Z are chosen to provide a codon for any amino acid residue except methionine and X', Y' and Z' are the appropriate complementary bases.
Claim 8
Dependent Claim
17 words
A method as recited in claim 1 wherein said synthetic double-stranded DNA codes for Insulin-like Growth Factor.
Claim 9
Independent Claim
132 words
A method of altering the nucleotide sequence of double-standard DNA within a structural gene encoding a protein, to prepare synthetic double-stranded DNA capable of expressing an analog of said protein, said method comprising the steps of: providing a synthetic double-stranded DNA comprising a sequence of codons capable of expressing the amino acids of said protein and providing a plurality of non native restriction endonuclease sites; digesting said synthetic double-stranded DNA with at least one restriction endonuclease having a cleavage recognition site within said DNA, whereby an intermediate DNA fragment is removed from a terminus of said synthetic DNA; and replacing said intermediate DNA fragment with a synthetic double-stranded oligonucleotide capable of being ligated to the terminus of said synthetic DNA, whereby the intermediate DNA fragment is replaced with said synthetic double-stranded oligonucleotide.
Claim 10
Independent Claim
132 words
A method of altering the nucleotide sequence of double-standard DNA within a structural gene encoding a protein, to prepare synthetic double-stranded DNA capable of expressing an analog of said protein, said method comprising the steps of: providing a synthetic double-stranded DNA comprising a sequence of codons capable of expressing the amino acids of said protein and providing a plurality of non native restriction endonuclease sites; digesting said synthetic double-stranded DNA with at least one restriction endonuclease having a cleavage recognition site within said DNA, whereby an intermediate DNA fragment is removed from a terminus of said synthetic DNA; and replacing said intermediate DNA fragment with a synthetic double-stranded oligonucleotide capable of being ligated to the terminus of said synthetic DNA, whereby the intermediate DNA fragment is replaced with said synthetic double-stranded oligonucleotide.
Claim 11
Dependent Claim
15 words
A method as recited in claim 8 wherein said double-stranded oligonuclease contains an initiation codon.
Claim 12
Dependent Claim
24 words
A method as recited in claim 8 wherein said synthetic double-stranded oligonucleotide comprises a nucleotide sequence distinct from that of said intermediate DNA fragment.
Claim 13
Dependent Claim
16 words
A method as recited in claim 8 wherein said synthetic double-stranded oligonucleotide contains a ternmination codon.
References Cited (Backward Citations)
GB2007676A
Cited
US4359535A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-12-18 | 1993-06-19 | 1993-12-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-12-18 | 1997-06-19 | 1997-12-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-12-18 | 2001-06-19 | 2001-12-19 | $7700.00 | Window Closed / Lapsed |