2 replies to this topic

[ikwana]

1. DNA sequencing requires a primer, which must be made with a specific sequence that can bind to an already known sequence on the molecule to be sequenced. in the case of directed sequencing, how do we know the sequence that the primer should be made to match? in the case of shotgun sequencing, how do we know the sequence of the primer to use?

2. in any sequencing project, errors occur in the actual synthesis of the DNA fragments and in the process of reading the fragment from the gel and base-calling. what is the main way of correcting those error in a large-scale sequenccing project?

3.what is the minimum coverage for shotgun contig?

4. why high coverage can be considered to represent a low error rate?

5.what reverse complement for? where is it to use?

Edited by ikwana, 15 August 2010 - 12:51 AM.

[mdfenko]

are these homework or test questions?

[ikwana]

mdfenko, on 15 August 2010 - 06:45 PM, said:

are these homework or test questions?

its homework.. i've already answered some of them.. so, i just wana compare my answer wit others..

these is my answer:

1. By using  the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing. To know the sequence that the primer should be made to match, primer pairs are  selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving  sequencing success rate, which currently exceeds 95% for exons, they have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. As a result, the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing.


4.
The coverage should occur between 5- and 8-fold  for maximum benefit and minimum overall cost.


is it correct?