General Category >> Help and How-to >> Digesting Created Fragment

Message started by R Smith on Feb 9th, 2012 at 12:23am

Title: Digesting Created Fragment
Post by R Smith on Feb 9th, 2012 at 12:23am
Using Cloning->Create Fragment, I have constructed a fragment (from annotated part of sequence) with 5' and 3' additions ("overhangs") containing restriction endonuclease sites (plus terminal 4 nts for enzyme foothold), but do not get a digest report after selecting the created fragment and using popup "Analyze->Find restriction sites."

Is it about missing qualifiers in the fragment annotation? Does the fragment not meet a digestion criterion?  What am I doing wrong?

Title: Re: Digesting Created Fragment
Post by Konstantin Okonechnikov on Feb 9th, 2012 at 4:17am
The fragment's "overhangs" (left and right ends) are only special-type of annotation's qualifier, they are not taken into account when searching for restriction sites or applying any other algorithm .

The purpose of these "overhang" qualifiers, along with "strand", "term" and "type",  is to provide information how the fragments can be "connected" during the construction of new molecule.

Let's say I see an interesting part of some sequence, which  I wish to "insert"  into some custom vector using EcoRi restriction site. However, this subsequence doesn't contain any restriction sites.

I create a fragment from the subsequence and add the sticky ends to it. Left sticky end will be AATT (direct strand), right sticky end TTAA (reverse strand)

Now I can use this fragment when constructing new molecules using "Cloning->Construct Molecule" dialog. (Screenshot 1)

Please let me know if you have more questions or my explanation is not detailed enough.

Recently we had some negative feedback from users about the misleading naming of items related to "cloning in silico" (see this bug for details), so we plan to improve this aspect. Any suggestions are welcome :)

ScreenHunter_03_Feb__08_21_56.gif (15 KB | )

Title: Re: Digesting Created Fragment
Post by R Smith on Feb 9th, 2012 at 5:09am
Okay, let me give you the real world problem so that you can see why just adding a single stranded DNA overhang to the termini won't work unless the annotated fragment double-stranded sequence itself is edited to include the extended site.

The user wants to create a fragment from the sequence coded by NCBI Nucleotide accession number CAA31046 (CD20).  The user wants to insert it into pUC19 (AccNum L09137) and decides to use the AvaI and SalI sites in the cloning multisite region of the vector, which means the user needs to put AvaI and SalI on the 5' and 3' ends of fragment CAA31046, which will be done in a PCR amplification.

The following sense (forward) and anti-sense (reverse) primer pairs are used to incorporate the sites:

sense:  5'-TGAATCCCGGG-[first 5' 15 or so nts of CAA31046]-3'
  where 5'-CCCGGG-3' is an AvaI site

anti-sense:  5'-AATAGTCGAC-[first 3' ?? nts of CAA31046]-3'
  which is  5'-GTCGACTATT-3' on the sense strand where 5'-GTCGAC-3' is a SalI site.

(The exact length of primers has been set so Tm is within 1-2 deg of each other and that 3' of primer is a C or G.)

So when the amplification is finished, the following amplicon is hopefully obtained:


and after the double digest the following is ready:

5'-CCGGG-CAA31046#####sense-G    -3'
3'-    C-CAA31046#antisense-CAGCT-5'

Since Create Fragment, then Digest won't work, the way I see it, the following must be done:

1.  The user must look at the double-stranded extensions to the annotated sequence from which a fragment must be created, perform the digest "on paper" and determine which of the extended parts remain double-stranded and which bases are single-stranded as a result of the digest (for enzyme not creating blunt ends).
2.  The double-stranded sequence to the fragment is then edited to include the double-stranded extensions.
3. Now Create Fragment can be used with the edited annotated sequence, and the overhangs representing the digest done.

My only question is if step 2 can be done (not tested by me yet).

If functionality can be created such that in the Create Fragment process, the user can specify 5' and 3' base pair (double-stranded) additions and then subsequently use the digestion function, that would seem useful.

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