Does anyone know why it is common to do the pcr for metagenomics in triplicate (same conditions and sample DNA) and then pool the products? Is it just to get enough DNA for the rest of protocol or is there some other reason?
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Hi,
I will give my explanation, I hope it matches other people's reason to do the PCR in triplicate. There several aspects and they all involve PCR-created biases.
As you have a collection of organisms, you don't expect every organisms' DNA to be amplified at the same cycle. Something that randomly starts to be amplified in the first cycles will be over-amplified in relation to other sequences. Other than that, DNA from less frequent organisms may be missed in one PCR because they are rare in the DNA fragments collection. Yet another reason is if you want to check for quantitative differences (which is always tricky). Because of the bias created by the exponential amplification, PCR-based sequencing can easily over/under-estimate the organisms frequency. The triplicate will buffer biases created in a single PCR. Having said all that, three is an arbitrary number of course, but it's the minimum number to help overcoming problems in one reaction.
Hope that helped.
Cheers.
Latest Articles
Collapse
-
by seqadmin
The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist on Modified Bases...-
Channel: Articles
Yesterday, 07:01 AM -
-
by seqadmin
Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...-
Channel: Articles
04-04-2024, 04:25 PM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 04-11-2024, 12:08 PM
|
0 responses
55 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
51 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
||
Started by seqadmin, 04-10-2024, 09:21 AM
|
0 responses
45 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 09:21 AM
|
||
Started by seqadmin, 04-04-2024, 09:00 AM
|
0 responses
55 views
0 likes
|
Last Post
by seqadmin
04-04-2024, 09:00 AM
|
Comment