Nader's Powerpoint (Converted to PDF)

Current library generation methods more or less follow this general set of steps.

1. Double stranded sample either cut up randomly or by some targeted approach.
2. The overhanging ends are either finished with polymerase (if on the right strand) or digested with exonuclease.
3. The blunt ended fragments are A tailed specifically on the 3' ends of the DNA.
4. Double stranded library adapters have overhanging T which allows ligation to blunt ends in a specific orientation.
   1. This orientation may be used to ensure single stranded samples

Since you are dealing with a short sequence you need a larger quantity of material to start due to loss in the gell size selection phase.

Capable of amplifying with high specificity with low (~2%) ribosomal RNA contamination.

1. First Strand cDNA synthesis
2. Second strand cDNA synthesis
3. SPIA Amplification
   1. RNAse H cuts up the RNA (SPIA) adapter because it is bound to the now DNA poly-A tail of the original RNA sample, and strips away some of one of the strands of cDNA randomly.
   2. Anneal back on an RNA SPIA primer (with attached RNA sequence to re-bind to the DNA poly-A tail for starting over from the first part of this step)
   3. Extend the cDNA sequence back out to its fullest extent with polymerase, adding the remainder of the partially degraded cDNA strand to the linearly growing pool of cDNA sample.
   4. Add RNAse H and continue with these steps until desired quantity is achieved.