Enzymatic Incorporation of

Transcription

1 The Modified ucleic Acid Experts Enzymatic Incorporation of Biotin-16-AA-dTs TriLink BioTechnologies, Inc. Research and Development Contributors: Joyclyn Yee, Stephanie erry, Michelle Mcamara, atasha aul

2 verview Biotin is a naturally occurring cofactor that binds very tightly to the tetrameric protein, avidin. This strong association between biotin and avidin has been used in a number of biotechnology applications, which include detection and isolation of a molecule of interest. ne approach for introducing biotin modifications into DA is by the enzymatic incorporation of nucleoside 5 -triphosphates that have been chemically modified with biotin.

3 utline Goal: Evaluate the enzymatic incorporation of Biotin-16-AA-dTs. Approach: Evaluate Biotin-16-AA-dT incorporation in primer extension experiments using either MMLV reverse transcriptase or Klenow(exo-) DA polymerase. Evaluate the incorporation of Biotin-16-AA-dTs in CR.

4 Chemical Structures of the Biotin-16-AA-dTs S 2 S Biotin-aminoallyl-2'-deoxyuridine- 5'-triphosphate Biotin-aminoallyl-2'-deoxycytidine- 5'-triphosphate

5 Single ucleotide Incorporation of Biotin-16-AAdTs by MMLV Reverse Transcriptase dat + + dct + + dgt + + dtt + + Biotin-16-AA-dCT + Biotin-16-AA-dUT + +1 extension product rimer = DA = RA 5 -FAM 3 AUCG 5 -FAM 3 GUCA All biotinylated dts are substrates for MMLV reverse transcriptase and produce an extension product with lower mobility. Experimental Conditions: 1x First strand synthesis buffer (50 mm Tris-Cl (p 25 o C), 50 mm KCl, 3.0 mm MgCl 2, 5 mm DTT), 5 -FAM-labeled primer (6.25 µm), RA template (10 µm), Ambion MMLV Reverse transcriptase (0.2 U/µL), 0.1 mm dt. Thermal cycling parameters: 42 o 20 min; 98 o 2 min.

6 Single ucleotide Incorporation of Biotin-16-AAdTs by Klenow(exo-) DA olymerase dat + + dct + + dgt + + dtt + + Biotin-16-AA-dCT + Biotin-16-AA-dUT + +1 extension product rimer = DA = RA 5 -FAM 3 ATCG 5 -FAM 3 GTCA All biotinylated dts are substrates for Klenow(exo-) DA polymerase and produce an extension product with lower mobility. Experimental Conditions: 1X EBuffer 2 (10 mm Tris-Cl (p 25 C), 50 mm acl, 10 mm MgCl 2, 1 mm DTT), 5 -FAMlabeled primer (10 µm), DA template (17 µm), ew England Biolabs Klenow(exo-) DA polymerase (0.025 U/µL), 20 µm dt. Thermal cycling parameters: 37 o 20 min; 72 o 20 min.

7 Summary of biotin-16-aa-dt Single ucleotide Incorporation Studies Relative incorporation of the modified nucleotide Biotin-16-AA-dUT Biotin-16-AA-dCT MMLV RT Klenow(exo-) Relative modified nucleotide incorporation = % n+1 extension product (modified dt) % n+1 extension product (natural dt) Relative to their natural counterpart, biotinylated dct and dut analogs are incorporated with ~50% lower efficiency at the conditions examined.

8 Incorporation of Biotinylated dts by Taq DA olymerase in CR % Biotin-16- AA-dCT* 100% 97.1% 96.8% 96.2% 95.6% 94.7% 93.3% 90.9% 85.7% 0% % Biotin-16- AA-dUT* 100% 88.9% 87.5% 85.7% 83.3% 80% 75% 66.6% 50% 0% *Total concentration of biotinylated + natural dt was maintained at 0.2 mm. The percentage of biotinylated dt was titrated between 0 and 100%. All biotinylated dts are substrates for Taq DA polymerase in CR. As the percentage of biotinylated nucleotide increases, a corresponding decrease in the mobility of the amplicon is observed. 100% biotinylated dt substitution causes complete reaction inhibition. Experimental Conditions: Control Lambda primers (0.2 mm), ew England Biolabs Taq DA olymerase (1 U/50 ml rxn), 0.2 mm dts (including Biotin and natural), 1x EB buffer (10 mm Tris-Cl (p 25 o C), 50 mm KCl, 1.5 mm MgCl 2, Lambda genomic DA (1 ng/50 µl rxn). rimer sequences: 5 -CCTGCTTCTGCCGCTTCACGA and 5 -TCCGGATAAAAACGTCGATGACATTTGC. Thermal cycling parameters: 95 o 2 min; [95 o 15 sec, 55 o 15 sec, 72 o 45 sec]25x; 72 o 5 min.

9 Incorporation of Biotinylated dts by Taq DA olymerase in CR For both biotinylated dts amplicon formation was possible at 89% substitution with biotin-16-aa-dut and at 97% substitution with biotin-16-aa-dct. Extent of biotinylated dt substitution that yields ~50% CR product formation: Biotin dct = ~92% Biotin dut = ~75% Details on data work-up: The percent product formation is the product yield, normalized to the yield with 100% unmodified dts. Data represent average amplicon yields from triplicate experiments, with the error bar representing the standard error of the mean.

10 Summary/utlook Biotin-16-AA-dCT and biotin-16-aa-dut can be readily incorporated by reverse transcriptases and DA polymerases in primer extension schemes. Biotinylated nucleotides are readily incorporated during CR amplification schemes. Biotin-16-AA-dCT can be substituted to a greater extent for its natural dt in CR without compromising amplicon yield. The strong performance of biotin-16-aa-dct in CR is of particular interest since many researchers commonly employ dut analogs in labeling schemes.