strand-specific cDNA - Page 2

TonyBrooks · 11-27-2013, 06:29 AM

You digest the second strand cDNA before PCR using an enzyme that cleaves wherever there is a uracil base (dUTP is used in place of dTTP in the second strand mastermix). This means when you do the PCR you are only amplifying from the first strand cDNA, hence all reads will from the same strand as the mRNA. The first strand cDNA will be complementary to the mRNA that produced it. The PCR product is still double-stranded

chaomeizhang · 11-27-2013, 06:58 AM

Appreciate...

my understanding is:
when you do PCR with two primers using the first strand cDNA as template, at first cycle it will use one of the primers (complimentary to the first strand cDNA) to generate a reverse complimentary strand that is actually is the second strand cDNA, and the following cycles will use the double strand DNA as template. How does the PCR only amplify the the first strand cDNA????

Thanks.

TonyBrooks · 11-27-2013, 07:11 AM

Both strands are created in the PCR, but by digesting the second strand you ensure that the P5 adapter is always on the 5' end of the mRNA and P7 is always on the 3' end. This means all reads from the sequencer will be the same sequence as the mRNA that made the library (Illumina read 1 are read P5->P7)

http://www.rna-seqblog.com/wp-conten...reparation.jpg

chaomeizhang · 11-27-2013, 07:34 AM

The point is that dUTP digestion of second strand is performed before PCR amplification. The following PCR cycles use the normal dNTP, there is no way to use the dUTP digestion to get rid of the second strand??? Thanks.

TonyBrooks · 11-27-2013, 08:09 AM

You remove the second strand before PCR. This means the template for the reaction is the first strand only. In an unstranded assay, both strands are used as template - so library is created from both strands.
The PCR amplicon is not digested - it is double stranded DNA.
Look at the jpg in the link. Sequencing is always in the blue to red direction.

AmitChaurasia · 07-01-2016, 04:05 AM

Thanks Tony & Chipper for your beautiful expression... I was also curious about the question asked by chaomeizhang. Thanks chaomeizhang.

chaomeizhang · 08-02-2017, 10:30 AM

Quote:

Originally Posted by Chipper

It is because you have two different adaptors (annealed in a Y-shape). Without dUTP you will get two different amplicons for each insert due to the Y-adaptors meaning that the first read can start from either end of the insert, but with dUTP you will only amplify one amplicon (which will be double stranded) so all reads from the same transcript will go in the same direction.

Thank you Chipper.

I would like to add some note to the explanation.

The enrichment PCR does regenerate the DNA strand which was degraded by UDG, but the adaptors on both sides of the strands can not hybridized with the lawn oligos on the flow cell, so these strands will be excluded during the cluster generation and eventually not be sequenced.

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11-27-2013, 07:11 AM	#23
TonyBrooks Senior Member Location: London Join Date: Jun 2009 Posts: 298	Both strands are created in the PCR, but by digesting the second strand you ensure that the P5 adapter is always on the 5' end of the mRNA and P7 is always on the 3' end. This means all reads from the sequencer will be the same sequence as the mRNA that made the library (Illumina read 1 are read P5->P7) http://www.rna-seqblog.com/wp-conten...reparation.jpg Last edited by TonyBrooks; 11-27-2013 at 07:12 AM. Reason: including image

07-01-2016, 04:05 AM	#26
AmitChaurasia Member Location: Delhi, INDIA Join Date: Jan 2013 Posts: 13	Thanks Tony & Chipper for your beautiful expression... I was also curious about the question asked by chaomeizhang. Thanks chaomeizhang. Last edited by AmitChaurasia; 07-01-2016 at 05:08 AM.

11-27-2013, 06:29 AM	#21
TonyBrooks Senior Member Location: London Join Date: Jun 2009 Posts: 298	You digest the second strand cDNA before PCR using an enzyme that cleaves wherever there is a uracil base (dUTP is used in place of dTTP in the second strand mastermix). This means when you do the PCR you are only amplifying from the first strand cDNA, hence all reads will from the same strand as the mRNA. The first strand cDNA will be complementary to the mRNA that produced it. The PCR product is still double-stranded

11-27-2013, 06:58 AM	#22
chaomeizhang Junior Member Location: Tampa, Florida Join Date: Aug 2011 Posts: 7	Appreciate... my understanding is: when you do PCR with two primers using the first strand cDNA as template, at first cycle it will use one of the primers (complimentary to the first strand cDNA) to generate a reverse complimentary strand that is actually is the second strand cDNA, and the following cycles will use the double strand DNA as template. How does the PCR only amplify the the first strand cDNA???? Thanks.

11-27-2013, 07:34 AM	#24
chaomeizhang Junior Member Location: Tampa, Florida Join Date: Aug 2011 Posts: 7	The point is that dUTP digestion of second strand is performed before PCR amplification. The following PCR cycles use the normal dNTP, there is no way to use the dUTP digestion to get rid of the second strand??? Thanks.

11-27-2013, 08:09 AM	#25
TonyBrooks Senior Member Location: London Join Date: Jun 2009 Posts: 298	You remove the second strand before PCR. This means the template for the reaction is the first strand only. In an unstranded assay, both strands are used as template - so library is created from both strands. The PCR amplicon is not digested - it is double stranded DNA. Look at the jpg in the link. Sequencing is always in the blue to red direction.