Dear Amera

Im a little confused about what exactly it is that you'd like advice on? Step 1 as you describe it above - is this to provide nested products for your second step or is it completely independent?

That said. best hybridization would be to ensure there is full-length target to bind to. Secondly, most enzymes that cut frequently (to digest sequence not of interest as you mentioned) could probably cut within your target region as well. It would most likely be best to digest and isolate your target fragment.

Essentially the options available to you are vast. I cant make a recommendation until I know what you're downstream application might be. Unless you're using RFLP or AFLP as a diagnostic tool? Which I would caution against.

Regards,

Nathaniel

Dear Dr. Nathaniel & Dr. Holger,

Thanks much for your useful comments, and I apologize if I improperly explained my points of concern.

First, I 'd like to confirm that both mentioned steps are done INDEPENDENTLY. Extracted DNA (of MTB-H37rv strain) will undergo two different paths:

1. Direct restriction digestion, then adding an oligo (20bp)-that is supposed to anneal with 16 complementary template sequences (100% matching) along the complete genome (4.200.000 bps),

2.  PCR is done, doing enzymatic digestion of amplicon. finally, adding the oligo.

***The reason why we try to skip the PCR step in the path#1 is to save cost and time, that might let the studied test more favorably applicable.

Second, I have prepared the full restriction mapping of MTB-H37rv. Oligo (20bps) is also BLASTed, and template matching locations are all identified (16 locations along MTB complete genome).

Please, let me clarify my queries through an  example of Bam HI enzyme mapping. Here is the report:

Cut frequency:1465

Cut site            Size of  Digested DNA(bp)        Oligo-matching site

999261                         17774                       888071-888090

1554980                      15929                       1539254-1539273

1986246                       2597                        1983852-1983871

1992318                       4834                        1992101-1992120

2361396                       2504                         2361179-2965987

2430910                       5771                         2425342-2425361

2545448                      1516                          2545231-2545250

2631011                       3922                         2630794-2630813

2782327                       2814                         2779716-2779735

2966185                      10185                       2965968-2965987

3120304                       6114                         3114393-3114412

3545254                      4222                         3545037-3545056

3546737                      1483                         3546520-3546539

3704580                      2228                         3704363-3704382

3794585                      5792                         3788996-3789015

3884854                     1258                          3884637-3545056

******Considering these data:

1.In your opinion, are these digested DNA pieces of optimum size that favors annealing with the oligo? (for example: at cute site #999261, oligo (20 bp) is supposed to hybridize with DNA template size 17774  bps)

2. Regarding cut frequency of the enzyme, suppose I have another enzyme that cuts 24,135 times ( ApeK I)  along the genomic sequence,. (and respects targeted sequences, too!). hence, giving more DNA pieces of smaller size. On this behalf, Do you think that cut frequency  is a relevant consideration in selecting the enzyme?

Thank you!

I am going to work directly on genomic DNA in the first option, as oligo will be added to extracted DNA not PCR product.

Another point is: from restriction enzyme mapping & oligo sequence BLAST, I can determine which enzymes shall not cut  within16 regions of interest. - please refer to above example of Bam HI.

Considering downstream application, I am going to add gold nanoparticles for detection after oligo hybridization step...

Do you think that I could skip enzymatic digestion step in case of genomic DNA work up?

Thank You!