A-base addition

seqgirl123 · 11-01-2009, 04:40 PM

In the polishing step for library preparation, there is a step using Klenow exo- polymerase and dATP, which will add one A-base to the 3' end of blunt ended DNA.

My question is how does only one A-base end up getting added onto the 3' end and not two or three?

seqgirl123 · 01-16-2010, 08:52 PM

does anyone know the answer to this?

shurjo · 01-16-2010, 10:07 PM

If recessed 3' ends are present, Klenow exo- fills them in and leaves a single base overhang in ~50% of cases. Given the blunt ends from the previous step in the Illumina protocol, it simply adds a single A to the 3' end.

seqAll · 01-17-2010, 03:33 AM

Thanks shurjo, that was very helpful!

Does it mean that, in the Illumina protocol, Klenow exo- adds a single A to ~50% of the blunted ends?

seqgirl123 · 01-17-2010, 01:49 PM

What are the other 50% cases about?

Sometimes it will not add at all correct? Will it ever add 2 or more A' bases?

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11-01-2009, 04:40 PM	#1
seqgirl123 Member Location: U.S Join Date: Oct 2008 Posts: 76	A-base addition In the polishing step for library preparation, there is a step using Klenow exo- polymerase and dATP, which will add one A-base to the 3' end of blunt ended DNA. My question is how does only one A-base end up getting added onto the 3' end and not two or three?

01-16-2010, 08:52 PM	#2
seqgirl123 Member Location: U.S Join Date: Oct 2008 Posts: 76	does anyone know the answer to this?

01-16-2010, 10:07 PM	#3
shurjo Senior Member Location: Rockville, MD Join Date: Jan 2009 Posts: 126	If recessed 3' ends are present, Klenow exo- fills them in and leaves a single base overhang in ~50% of cases. Given the blunt ends from the previous step in the Illumina protocol, it simply adds a single A to the 3' end.

01-17-2010, 03:33 AM	#4
seqAll Member Location: China Join Date: Nov 2009 Posts: 21	Thanks shurjo, that was very helpful! Does it mean that, in the Illumina protocol, Klenow exo- adds a single A to ~50% of the blunted ends?

01-17-2010, 01:49 PM	#5
seqgirl123 Member Location: U.S Join Date: Oct 2008 Posts: 76	What are the other 50% cases about? Sometimes it will not add at all correct? Will it ever add 2 or more A' bases?