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--- lecture_notes:04-14-2010 [2010/04/16 19:54]
galt
+++ lecture_notes:04-14-2010 [2010/04/17 05:59]
jstjohn
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-Library Construction
+===== 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.

Banana Slug Genomics

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