Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunat... more Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensi- tivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly
Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's ... more Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were adm...
Background: All states require some kind of testing for newborns, but the policies are far from s... more Background: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expan- sion of newborn screening. We describe the develop- ment and technical evaluation of a multiplex platform
Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunat... more Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensi- tivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target. Methods: We developed a novel real-time PCR technol- ogy that uses universal energy transfer probes con- structed from An Expanded Genetic Information Sys- tem (AEGIS) for both quantification and genotyping analyses. Results: RNA quantification by reverse transcription- PCR was linear over four orders of magnitude for the simultaneous analysis of -actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was per- formed by endpoint ...
This unit describes a simple and efficient synthesis of the phosphoramidite derivative of N(1)-me... more This unit describes a simple and efficient synthesis of the phosphoramidite derivative of N(1)-methyl-2'-deoxyadenosine from 2'-deoxyadenosine. The synthesis starts with the monomethoxytritylation of 2'-deoxyadenosine followed by methylation of 5'-O-protected nucleoside at N-1. Subsequent N-chloroacetylation leads to N(6)-chloroacetyl-N(1)-methyl-5'-O-(p-anisyldiphenylmethyl)-2'-deoxyadenosine, which is finally converted to its 3' phosphoramidite derivative. This phosphoramidite is used to incorporate N(1)-methyl-2'-deoxyadenosine into synthetic oligonucleotides. N-Chloroacetyl protection and controlled anhydrous deprotection conditions are used to avoid the Dimroth rearrangement.
Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunat... more Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensi- tivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly
Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's ... more Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were adm...
Background: All states require some kind of testing for newborns, but the policies are far from s... more Background: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expan- sion of newborn screening. We describe the develop- ment and technical evaluation of a multiplex platform
Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunat... more Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensi- tivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target. Methods: We developed a novel real-time PCR technol- ogy that uses universal energy transfer probes con- structed from An Expanded Genetic Information Sys- tem (AEGIS) for both quantification and genotyping analyses. Results: RNA quantification by reverse transcription- PCR was linear over four orders of magnitude for the simultaneous analysis of -actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was per- formed by endpoint ...
This unit describes a simple and efficient synthesis of the phosphoramidite derivative of N(1)-me... more This unit describes a simple and efficient synthesis of the phosphoramidite derivative of N(1)-methyl-2'-deoxyadenosine from 2'-deoxyadenosine. The synthesis starts with the monomethoxytritylation of 2'-deoxyadenosine followed by methylation of 5'-O-protected nucleoside at N-1. Subsequent N-chloroacetylation leads to N(6)-chloroacetyl-N(1)-methyl-5'-O-(p-anisyldiphenylmethyl)-2'-deoxyadenosine, which is finally converted to its 3' phosphoramidite derivative. This phosphoramidite is used to incorporate N(1)-methyl-2'-deoxyadenosine into synthetic oligonucleotides. N-Chloroacetyl protection and controlled anhydrous deprotection conditions are used to avoid the Dimroth rearrangement.
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