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--- lecture_notes:mar31 [2010/04/01 02:56]
jstjohn
+++ lecture_notes:mar31 [2010/04/01 03:04]
jstjohn
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 == Pyrosequencing ==
+When a nucleotide is added to the template chain by a polymerase, a PPi is released which is converted to an ATP by one enzyme. In the presence of ATP another enzyme, luciferase, releases light. You record when the light was released (ie which nucleotide was added to the plate at that time) and also the intensity of the light. The light intensity tells you how many nucleotides were added in a row.
+After each nucleotide is added, you must wash away the nucleotides and start fresh before adding the next nucleotide and recording which positions on the plate light up in that case.
+There is a problem with multiple A's because of the modified nucleotides which are needed for this reaction. FIXME
+You must get the ammount of template on the beads just right. Must be all the same type. If too much template is present the read length is shortened (not sure why). If too few template is on the beads then the washing step will take away a significant amount of template and you won't have enough of a signal.
+The packing beads that are placed around the bead which holds the template strands in the individual wells on the plate are what hold the various enzymes (other than the polymerase) needed for the reaction.
 == solid ==

Banana Slug Genomics