Generation and application of standardized universal libraries
View Patent ↗This invention provides quantitative, systematic and standardized High Throughput Screening (HTS) methods capable to the identification of both known and unknown sequences without prior knowledge of the sequences of interest. Either coding or non-coding sequences could be targeted, identified and analyzed systematically and respectively at genomic, transcriptional and translational levels with those methods. The genetic algorithms for sequence deducing and standardized universal libraries constructing are provided. The genetic algorithms are 61.sup.(n−m), 61.sup.n, 64.sup.(n−m), 64.sup.n., 20.sup.(n−m) and 20.sup.n. Applications of the standardized universal libraries include gene expression profiling, signature sequence identification and sequence determination by PCR, cloning, dot-blot hybridization, ELISA, DNA and Peptide Arrays.
1. A method of preparing a cDNA library comprising the following method steps:
a. preparing an oligonucleotide library comprising all possible combinations of 61 codons that encode the 20 essential amino acids and each oligonucleotide has an orientation sequence wherein the orientation sequence is selected from the group consisting of a 5′ end start codon, a 3′ end stop codon, a 3′ end antisense start codon, a 5′ end antisense stop codon, a 5′ end two codon restriction endonuclease recognition sequence, a 3′ end two codon restriction endonuclease recognition sequence, a 5′ end antisense two codon restriction endonuclease recognition sequence, and a 3′ end antisense two codon restriction endonuclease recognition sequence and wherein the length of each oligonucleotide is a multiple of three based on the codon length,
b. mixing an mRNA sample containing a plurality of different mRNA molecules with the oligonucleotide library of method step a wherein the orientation sequence orients each oligonucleotide,
c. incubating the mixture of method step b with oligo-d(T)s and reagents needed for reverse transcription under conditions suitable for mRNA reverse transcription thereby producing first strands of a cDNA library,
d. incubating the first strands of a cDNA library of method step c with reagents needed for cDNA synthesis under conditions suitable for cDNA synthesis thereby producing a double stranded cDNA library,
e. amplifying the double stranded cDNA library of method step d via polymerase chain reaction, and
f. isolating each cDNA of method step e.
2. The method of claim 1 , wherein said oligonucleotides are grouped according to GC content.
3. The method of claim 1 , wherein said oligonucleotide library comprises at least 80% of said oligonucleotides.
4. The method of claim 1 , wherein each of said cDNA is immobilized to or on a suitable solid support in a set of each of said cDNA at a specific discrete position to form an array, the said set comprising at least two copies of said cDNA, the said array comprising at least two said sets.