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The method of any one of the preceding claims wherein only failure sequences of the targeted double-stranded DNA sequence equivalents are present in the broths.ħ. The method of claim 3 wherein the first two cycles of the polymerase chain reaction is carried out over a period of at least 30 minutes.Ħ. The method of claim 3 wherein the first two cycles of the polymerase chain reaction are carried out over a period of greater than 1 minute.ĥ. The method of claim 1 or 2 wherein the initial cycles of the polymerase chain reaction are carried out over a period of time to allow hybridization of failure sequences.Ĥ.
PCR 800 FFF ERROR SERIAL
A method of making targeted double-stranded DNA sequences having more than 400 base pairs characterized by the steps of: A) preparing a series of first and second synthesis broths, each comprising i) DNA oligonucleotides of a defined nucleotide length and failure sequences thereof or ii) the failure sequences only wherein a) the first broth of each serial member of the series comprises DNA oligonucleotides equivalent to a defined portion of the top strand or failure sequences thereof of the targeted double-stranded DNA sequence b) the second broth in each serial member of the series comprises DNA oligonucleotides equivalent to a defined portion of the bottom strand or failure sequences thereof of the targeted double-stranded DNA sequence and c) the entire series, taken together, is equivalent to the entire targeted double-stranded DNA sequence B) separately mixing the first and second synthesis broths of each member of the series together C) adding to each mixture of the series DNA oligonucleotide primers that are complementary to the 3 min ends of the DNA oligonucleotides in each mixture D) applying polymerase chain reaction procedures to each mixture thereby amplifying exponentially the concentration of the targeted double-stranded DNA sequence equivalents in each mixture E) isolating the targeted double-stranded DNA sequence equivalents from each mixture in the series F) cloning each of the equivalents in a cloning vector having a phage origin of replication G) sequencing each equivalent to identify any mutations H) repairing any mutations by site-directed mutagenesis I) removing each equivalent of the series from the cloning vector J) ligating the entire series of equivalents together to form the targeted double-stranded DNA sequence having more than 400 base pairs.ģ.
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A method of making targeted double-stranded DNA sequences characterized by the steps of: A) preparing first and second synthesis broths, seach comprising i) DNA oligonucleotides of a defined nucleotide length and failure sequences thereof or ii) the failure sequences only wherein the first synthesis broth comprises DNA oligonucleotides that include the top strand or failure sequences thereof of the targeted double-stranded DNA sequence and the second synthesis broth comprises DNA oligonucleotides that include the bottom strand or failure sequences thereof of the targeted DNA sequence B) mixing the first and second synthesis broths together C) adding DNA oligonucleotide primers that are complementary to the 3 min ends of the DNA oligonucleotides of A) D) applying polymerase chain reaction (PCR) procedures to the admixture of C) thereby amplifying exponentially the concentration of the target double-stranded DNA sequence E) isolating the target double-stranded DNA sequence F) cloning the target double-stranded DNA sequence in a cloning vector having a phage origin of replication G) sequencing the target double-stranded DNA sequence to identify mutations and H) repairing the mutations by site-directed mutagenesis.Ģ.