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===== Library Construction ====== {{:lecture_notes:235_library_construction.pdf|Nader's Powerpoint}} (Converted to PDF) ==== General Overview ==== Current library generation methods more or less follow this general set of steps. - Double stranded sample either cut up randomly or by some targeted approach. - The overhanging ends are either finished with polymerase (if on the right strand) or digested with exonuclease. - The blunt ended fragments are A tailed specifically on the 3' ends of the DNA. - Double stranded library adapters have overhanging T which allows ligation to blunt ends in a specific orientation. - This orientation may be used to insure single stranded samples ==== 454 Specific Library Construction ==== ==== SOLiD Specific Library Construction ==== 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. === Mate Pair Library === ==== Illumina Specific LIbrary Construction ==== === Paired End Library === ==== Targeted RNA Amplification ==== Capable of amplifying with high specificity with low (~2%) ribosomal RNA contamination. - First Strand cDNA synthesis - Second strand cDNA synthesis - SPIA Amplification - 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. - 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) - 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. - Add RNAse H and continue with these steps until desired quantity is achieved.
