UGENE Forum - enhanced primer search

Jan 27^th, 2016 at 6:55pm

Nex Offline
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UK

Posts: 6

Hello, thanks (as always) for the hard work on Ugene, and for carrying on!

I would like to make a suggestion for an especial case in searches: when you search for primer binding sites in a sequence.

Currently, a search can be relaxed down by setting a % matching. However, in the case of primers this gives information that can be filtered. Polymerisation in PCR starts at the 3' end of a primer, so matching the 3' is critical whilst matching the 5' end can be irrelevant (e.g. in a long sequence, the 5' end can be just hanging free, but the primer attaches to the sequence by the 3' end and polymerisation starts).

The matching algorithm can be as complicated as you wish, but I think that in order of preference:
- I would give more weigh to the 3' end 1to5 > 6to10 > 10to15 bases, and would add a tick to consider irrelevant the 20+ bases towards the 5' end, or when starting from the 3' end a threshold Tm is reached.
- G and C at the 3' end have more weigh than A and T (due to melting temperature)
- melting temperature calculations can be made, to apply thresholds (e.g. discard melting temperatures below 45 degrees).
- a combination of melting temperature and matching (number of bases matched, giving more weight to bases clustered closer to the 3' end) can be scored, so the primers are given in a meaningful order (primers with more matching bases at the 3' end can start polymerisation more likely. But e.g. a primer with no matches in the first 1 bases at the 3' is will not polymerise unless reaction conditions are relaxed, and a primer with 2 unmatched bases at the 3' end will be extremely unlikely to polymerise in standard conditions).
- a results window can be provided, visually showing the matching of the primers like an alignment, so the user can visually assess (matching algorithms can be initially simpler then) and select primer binding sites to be added to the project.
- It would be nice to show 5' unmatched binding with hanging ends like Snapgene does.

Hope the suggestions help, any comments appreciated.
All the best!

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Reply #1 - Sep 19^th, 2016 at 6:10pm

ClayAnderson2 Offline
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Posts: 5

thank you

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Reply #2 - Dec 19^th, 2016 at 3:34pm

Doucan Offline
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Posts: 1

Good suggestion.

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Reply #3 - Apr 13^th, 2019 at 3:06am

Jamesym Offline
YaBB Newbies

Posts: 1

Nex wrote on Jan 27^th, 2016 at 6:55pm:

Hello, thanks (as always) for the hard work on Ugene, and for carrying on!

I would like to make a suggestion for an especial case in searches: when you search for primer binding sites in a sequence.

Currently, a search can be relaxed down by setting a % matching. However, in the case of primers this gives information that can be filtered. Polymerisation in PCR starts at the 3' end of a primer, so matching the 3' is critical whilst matching the 5' end can be irrelevant (e.g. in a long sequence, the 5' end can be just hanging free, but the primer attaches to the sequence by the 3' end and polymerisation starts).

The matching algorithm can be as complicated as you wish, but I think that in order of preference:
- I would give more weigh to the 3' end 1to5 > 6to10 > 10to15 bases, and would add a tick to consider irrelevant the 20+ bases towards the 5' end, or when starting from the 3' end a threshold Tm is reached.
- G and C at the 3' end have more weigh than A and T (due to melting temperature)
- melting temperature calculations can be made, to apply thresholds (e.g. discard melting temperatures below 45 degrees).
- a combination of melting temperature and matching (number of bases matched, giving more weight to bases clustered closer to the 3' end) can be scored, so the primers are given in a meaningful order (primers with more matching bases at the 3' end can start polymerisation more likely. But e.g. a primer with no matches in the first 1 bases at the 3' is will not polymerise unless reaction conditions are relaxed, and a primer with 2 unmatched bases at the 3' end will be extremely unlikely to polymerise in standard conditions).
- a results window can be provided, visually showing the matching of the primers like an alignment, so the user can visually assess (matching algorithms can be initially simpler then) and select primer binding sites to be added to the project.
- It would be nice to show 5' unmatched binding with hanging ends like Snapgene does.

Hope the suggestions help, any comments appreciated.
All the best!

Thank you so much it is very informative and i am glad to be here and get valuable solutions

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Reply #4 - Oct 16^th, 2020 at 5:55pm

MichaelGoins Offline
YaBB Newbies

Posts: 1

Quote:

Good suggestion.

Could you please suggest me or guide me what he say ?

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