NSCR
Nascent Strand Capture and Release
NSCR isolates and sequences the origin of DNA replication by capturing short nascent strands (SNS), which are single-stranded RNA-DNA chimeras (Kunnev et al., 2015). In short, NSCR captures and purifies RNA-DNA chimeric SNS, and releases the DNA part by cutting the strand at the end of the initial RNA primer section.
First, gDNA is denatured, size-selected through a sucrose gradient for 400_2000 bp fragments, and 5ê-biotinylated. The biotinylated fragments are isolated by streptavidin pull-down, purifying both SNS and gDNA strands. Next, RNase I is used to separate the DNA from the initial RNA primer section of the single-stranded RNA-DNA chimera. This process leaves RNA primers and gDNA on the streptavidin beads. The released nascent DNA strands are amplified and sequenced. This method is an improvement over widely used SNS purification methods using BrdU and lambda exonuclease (_-exo).
Advantages:
- Maps genome-wide DNA replication origins
- Higher discovery of nascent strands than _-exo_based methods (2231 vs 3922 peaks)
- RNase I cleaves with minimal sequence specificity
- Can be used for systems that use small RNA primers to produce RNA-DNA chimeras
Disadvantages:
- Less than 10% purification yield (1_2 ng of SNS from 1 mg total DNA)
- False reads may arise due to misincorporation of ribonucleotides into the DNA portion of the SNS
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
None available yet
References:
Kunnev D., Freeland A., Qin M., Wang J. and Pruitt S. C. Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR). J Biol Methods. 2015;2:
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History: NSCR
Revision by sbrumpton on 2017-06-21 09:06:27 - Show/Hide
Nascent Strand Capture and Release
NSCR isolates and sequences the origin of DNA replication by capturing short nascent strands (SNS), which are single-stranded RNA-DNA chimeras (Kunnev et al., 2015). In short, NSCR captures and purifies RNA-DNA chimeric SNS, and releases the DNA part by cutting the strand at the end of the initial RNA primer section.
First, gDNA is denatured, size-selected through a sucrose gradient for 400_2000 bp fragments, and 5ê-biotinylated. The biotinylated fragments are isolated by streptavidin pull-down, purifying both SNS and gDNA strands. Next, RNase I is used to separate the DNA from the initial RNA primer section of the single-stranded RNA-DNA chimera. This process leaves RNA primers and gDNA on the streptavidin beads. The released nascent DNA strands are amplified and sequenced. This method is an improvement over widely used SNS purification methods using BrdU and lambda exonuclease (_-exo).
Advantages:- Maps genome-wide DNA replication origins
- Higher discovery of nascent strands than _-exo_based methods (2231 vs 3922 peaks)
- RNase I cleaves with minimal sequence specificity
- Can be used for systems that use small RNA primers to produce RNA-DNA chimeras
Disadvantages:- Less than 10% purification yield (1_2 ng of SNS from 1 mg total DNA)
- False reads may arise due to misincorporation of ribonucleotides into the DNA portion of the SNS
Reagents:Illumina Library prep and Array Kit SelectorReviews:None available yet
References:Kunnev D., Freeland A., Qin M., Wang J. and Pruitt S. C. Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR). J Biol Methods. 2015;2: