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Codon Optimization. Codon Optimization. Codon Usage Bias: Variable frequency of synonomous codons . Codon preferences reflect natural selection in organisms for translational optimization. Optimal codons allow faster translation rates, thus highly expressed genes are coded by these. - PowerPoint PPT Presentation
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Codon Optimization
Codon Usage Bias: Variable frequency of synonomous codons.
Codon preferences reflect natural selection in organisms for translational optimization.
Optimal codons allow faster translation rates, thus highly expressed genes are coded by these.
Codon optimization can be used to produce a synthetic version of a foreign gene for more efficient expression.
Codon Optimization
Codon Usage of HIV vs. Human
Expression Profile of Synthetic vs. Wildtype gp120
HIV Codons Decrease Efficiency of Thy-1 Expression
-Thy-1 is a highly expressed cell-surface protein.
-Replacing codons of Thy-1 with HIV envelope codons significantly decreases expression.
A: Control: Empty vector.
B: Native GFP from Aequorea victoria.
C: Mammalian codon optimized GFP.
D: Mammalian codon optimized GFP + S65T replacement.
Codon Optimization of GFP
E. Coli vs. Human Codon Usage
Codon Optimization: Human to Bacteria
Codon Optimization: Human to Bacteria
Codon Optimization: Human to Bacteria
Codon Optimization: Human to Bacteria
Codon Optimization: Human to Bacteria
Molecular biology technique used to amplify specific nucleotide sequences.
Developed by Kary Mullis in 1983.Takes advantage of Taq polymerase from
thermophile Thermus aquaticus.Taq is a heat-resistant enzyme that can
function in temperatures above 70°C.
Polymerase Chain Reaction
Polymerase Chain Reaction
Polymerase Chain Reaction
WaterTaq Buffer containing divalent and monovalent
cations – Provides suitable chemical environment for optimal activity of DNA polymerase (pH, [salt], co-factors,etc.)
DNA template containing target region to be amplified.
Sense and anti-sense primers.Deoxynucleotide triphosphates (dNTPs), the
building-blocks for synthesis of new DNA.Taq polymerase – has optimal activity around 70°C.
Components of PCR
NCBISearch through GENE databases.Enter the name of the gene of interest.Search for Homo sapien or E. coli and select
the gene.Scroll down to mRNA and proteins and select.Copy the “translation”
EncorbioSelect the organism that the gene will be
codon optimized to.Paste the amino acid sequence into the top box.Generate the coding sequence.
Homework Assignment
Amino Acid Sequence MKSGSGGGSPTSLWGLLFLSAALSLWPTSGEICGPGIDIRNDYQQLKRLENCTVIEGYLHILLISKAEDYRSYRFPKLTVITEYLLLFRVAGLESLGDLFPNLTVIRGWKLFYNYALVIFEMTNLKDIGLYN
LRNITRGAIRIEKNADLCYLSTVDWSLILDAVSNNYIVGNKPPKECGDLCPGTMEEKPMCEKTTINNEYNYRCWTTNRCQKMCPSTCGKRACTENNECCHPECLGSCSAPDNDTACVACRHYYYAGVCVPACPPNTYRFEGWRCVDRDFCANILSAESSDSEGFVIHDGECMQECPSGFIRNGSQSMYCIPCEGPCPKVCEEEKKTKTIDSVTSAQMLQGCTIFKGNLLINIRRGNNIASELENFMGLIEVVTGYVKIRHSHALVSLSFLKNLRLILGEEQLEGNYSFYVLDNQNLQQLWDWDHRNLTIKAGKMYFAFNPKLCVSEIYRMEEVTGTKGRQSKGDINTRNNGERASCESDVLHFTSTTTSKNRIIITWHRYRPPDYRDLISFTVYYKEAPFKNVTEYDGQDACGSNSWNMVDVDLPPNKDVEPGILLHGLKPWTQYAVYVKAVTLTMVENDHIRGAKSEILYIRTNASVPSIPLDVLSASNSSSQLIVKWNPPSLPNGNLSYYIVRWQRQPQDGYLYRHNYCSKDKIPIRKYADGTIDIEEVTENPKTEVCGGEKGPCCACPKTEAEKQAEKEEAEYRKVFENFLHNSIFVPRPERKRRDVMQVANTTMSSRSRNTTAADTYNITDPEELETEYPFFESRVDNKERTVISNLRPFTLYRIDIHSCNHEAEKLGCSASNFVFARTMPAEGADDIPGPVTWEPRPENSIFLKWPEPENPNGLILMYEIKYGSQVEDQRECVSRQEYRKYGGAKLNRLNPGNYTARIQATSLSGNGSWTDPVFFYVQAKTGYENFIHLIIALPVAVLLIVGGLVIMLYVFHRKRNNSRLGNGVLYASVNPEYFSAADVYVPDEWEVAREKITMSRELGQGSFGMVYEGVAKGVVKDEPETRVAIKTVNEAASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQPTLVIMELMTRGDLKSYLRSLRPEMENNPVLAPPSLSKMIQMAGEIADGMAYLNANKFVHRDLAARNCMVAEDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTYSDVWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEIISSIKEEMEPGFREVSFYYSEENKLPEPEELDLEPENMESVPLDPSASSSSLPLPDRHSGHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALPLPQSSTC
Codon Optimized Sequence ATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCGGCCCGGGCATTGATATTCGCAACGATTATCAGCAGCTGA
AACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGGCGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCTTTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGGAACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACTGGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACGCGCGCTGCCGCTGCCGCAGAGCAGCACCTGC
Example: Insulin-like Growth Factor 1 Receptor (IGF1R)
Add EcoRI (GAATTC) at 5’ end and BamHI (GGATCC) at 3’ end. GAATTCATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCGGC
CCGGGCATTGATATTCGCAACGATTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGGCGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCTTTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGGAACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACTGGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACGCGCGCTGCCGCTGCCGCAGAGCAGCACCTGCGGATCC
Example: Insulin-like Growth Factor 1 Receptor (IGF1R)
Add EcoRI (GAATTC) at 5’ end and BamHI (GGATCC) at 3’ end. GAATTCATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCGGC
CCGGGCATTGATATTCGCAACGATTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGGCGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCTTTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGGAACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACTGGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACGCGCGCTGCCGCTGCCGCAGAGCAGCACCTGCGGATCC
Example: Insulin-like Growth Factor 1 Receptor (IGF1R)
~5’ End5’GAATTCATGAAAAGCGGCAGCGGCGGCGGC3’3’CTTAAGTACTTTTCGCCGTCGCCGCCGCCG5’~*You need an available 3’OH, so for this end you
make a primer against the antisense strand.5’GAATTCATGAAAAGCGGCAGCGGCGGCGGC3’
5’GAATTCATGAAAAGCGGCAGCGGC3’OH3’CTTAAGTACTTTTCGCCGTCGCCGCCGCCG5’
Designing Amplification Oligos
~3’ End5’TGCCGCTGCCGCAGAGCAGCACCTGCGGATCC3’3’ACGGCGACGGCGTCTCGTCGTGGACGCCTAGG5’~Again, you need a 3’OH so this time make a primer
against the sense strand.5’TGCCGCTGCCGCAGAGCAGCACCTGCGGATCC3’ HO3’GGCGTCTCGTCGTGGACGCCTAGG5’
3’ACGGCGACGGCGTCTCGTCGTGGACGCCTAGG5’
Designing Amplification Oligos
Primers need to be anywhere from 18-25 nucleotides.
Don’t include the restriction site or Kozak sequence as part of your 18-25 nucleotides for the primer.
Should contain at least 50% GC composition.
Must flank the entire CODING sequence.
Designing Amplification Oligos