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Showing posts from October, 2012

Paper of the week - explaining the stability of ncRNAs in the cell

3' Polyadenylation is a key mechanism whereby mRNAs are stabilised and made ready for protein translation. One of the mysteries of molecular biology of late is how long non coding RNA (lncRNA) stability is achieved given that these molecules don't have a polyadenylation signal. Wilusz et al published a paper in Genes and Development predicting that MALAT1 is protected from 3' to 5' exonuclease activity by an RNA triple helix structure. The researchers used molecular modeling to resolve that the 3' terminus is neatly tied into a triple helix and thus likely protected from degradation. This was confirmed by mutagenesis, showing that altering bases in these regions led to a reduction in transcript stability.

MALAT1 is transcribed to form a ~6.7 kb lncRNA which is abundant in the nucleus, and also producing a small tRNA like transcript which is processed into a mature 61 nt hairpin localised to the cytosol. Both transcripts are dependant on RNase P, a ribozyme which is …

DNA library preparation for next-generation sequencing

Library preparation is a process in which we modify DNA into a form that it is compatible for high throughput sequencing, and is becoming a key molecular biology technique. While there are an amazing variety of different library preparation methods available, I thought I'd start the the blog with a description of the classic method:
- Shearing/fragmentation
- End Repair
- DNA clean-up
- A tailing
- Adaptor ligation
- Size selection
- Amplification
- Quality control

Shearing/Fragmentation
The DNA needs to be in a size range that is compatible with the sequencing platform. The most commonly used sequencing platforms require DNA construct in the range of 300-500 bp, although this depends on the specific platform and the application. Fragmentation can be done by mechanical disruption through sonication like we do in our lab, but can also be done with a nebuliser or with enzymatic fragmentation. Our thoughts are that sonication/nebuliser has a lesser degree of sequence specificity bias …

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Welcome to Genome Spot.

Genome Spot is aimed at providing practical tips in applications of genomics and bioinformatics. From time to time, we'll also be discussing general news and recent articles.

About the author: Mark Ziemann is a researcher at Baker IDI Melbourne currently working in the field of epigenomics with a focus on human disease.

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