Using BLAST to check primers - (Aug/11/2009 )

HomeBrew on Aug 12 2009, 09:48 AM said:

Ahhhh, well that makes things different for me. So when I do my PCR should I make my annealing temperatures close to those of the primer Tm without the added 5' sequences? For example, if my primers without the added 5' sequence have a Tm of 65 and the primers with the added 5' sequence have a Tm of 82, I would use a temperature closer to 65 for my annealing temps? I have been designing my primers by eye, I tried using the Primer BLAST, which I believe is powered by Primer3, and I would get primers which ended up all throughout the gene rather than on the ends of the genes where I need them. Maybe I'll give it another go then. Thanks again for your patience and answers! It's much appreciated.

The gene I'm trying to clone out BTW is gene encoding for gyrA, which are base pairs 7302-9818 of the H37Rv genome. Apparently I have some more work to do here! Something that seems so simple and I'm completely goofing it up!

NYYFan on Aug 12 2009, 02:51 PM said:

Correct. Assuming you must begin your forward primer at base 1 (the "A" of the ATG start codon) and end it at base 2517 (the final "A" of the TAA stop codon), and using the sequence found here, about the best you can do is something like:

OLIGO start len tm gc% any 3' seq
LEFT PRIMER 1 20 62.18 55.00 5.00 3.00 atgacagacacgacgttgcc
RIGHT PRIMER 2517 20 61.02 50.00 4.00 1.00 ttaattgcccgtctggtctg

Product Size: 2517

1 atgacagacacgacgttgccgcctgacgactcgctcgaccggatcgaaccggttgacatc
>>>>>>>>>>>>>>>>>>>>
.
.
.

2461 gaaagtggcgacgataatgccgtggacgccaacggcgcagaccagacgggcaattaa
<<<<<<<<<<<<<<<<<<<<

NYYFan on Aug 12 2009, 02:51 PM said:

The easiest way I've found to pick primers when you must have them begin at particular bases is to use the Primer3Plus interface to Primer3 (see an example here). You paste your sequence in the box, select "Cloning" from the Task box in the upper left, and identify where your primers must begin using "{" to mark the position where the forward primer must start and "}" for where the reverse primer must start (without the quotes, of course -- e.g. the sequence in the box for this example would look like {atgaca...aattaa}), set your parameters in the General Settings tab -- for example, I usually set minimum primer length to 20, optimal to 24, and max to 30, but you can leave these at the defaults if you like), and click on the Pick Primers button.

The program will perhaps complain about minor defects in the primer (it yells about the 3' stability of the forward primer above, for example), but since you must have the primers start at a particular base your choices are minimal, and (according to Primer3) this is the best set of primers possible, defects and all (I've never had a problem with some of the errors Primer3 yells about...).

NYYFan on Aug 12 2009, 02:51 PM said:

No problem. Someone showed me how to do it long ago, and I'm paying them back -- even if I'm a Red Sox fan and can see Fenway out my lab window....

You design your PCR program around the Tm's shown above. Don't forget to append the sequences you need (in 5'->3' direction) to the primers before you order them!

BTW, when you BLAST these primers against Mycobacterium tuberculosis H37Rv, you get:

Primer pair 1
Sequence (5'->3') Length Tm GC%
Forward primer ATGACAGACACGACGTTGCC 20 55.26 55.00%
Reverse primer TTAATTGCCCGTCTGGTCTG 20 52.10 50.00%


>NC_000962.2 Mycobacterium tuberculosis H37Rv, complete genome

product length = 2517
Features associated with this product:
DNA gyrase subunit A

Forward primer 1 ATGACAGACACGACGTTGCC 20
Template 7302 .................... 7321

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 9818 .................... 9799


product length = 2253
Features associated with this product:
hydrolase

hypothetical protein

Forward primer 1 ATGACAGACACGACGTTGCC 20
Template 3109070 CG...G.C.G.C........ 3109089

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 3111322 GACC...G..A........T 3111303


product length = 1528
Features associated with this product:
3-ketoacyl-(acyl-carrier-protein) reductase

acetyl-CoA acetyltransferase

Forward primer 1 ATGACAGACACGACGTTGCC 20
Template 290690 CA....CGA.T......... 290709

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 292217 GCG.C.CG...C........ 292198


product length = 5800
Features associated with this product:
putative acetyl-CoA carboxylase biotin carboxyl carrier p...

anti-sigma factor

Forward primer 1 ATGACAGACACGACGTTGCC 20
Template 3597669 .GC.GCAC........C... 3597688

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 3603468 ACC.GC.....GT....... 3603449


product length = 5019
Features associated with this product:
hypothetical protein

methanol dehydrogenase transcriptional regulatory protein...

Forward primer 1 ATGACAGACACGACGTTGCC 20
Template 3531859 .CAG..AC........G... 3531878

Forward primer 1 ATGACAG-ACACGACGTTGCC 20
Template 3536877 -.CT.G.T...A......... 3536858


product length = 4627
Features associated with this product:
hypothetical protein

hypothetical protein

Forward primer 1 ATGACAGACACGACGTTGCC 20
Template 3114295 TCAT..AT........C... 3114314

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 3118921 GGGTAGC....G........ 3118902


product length = 3005
Features associated with this product:
acyltransferase

transcriptional regulatory protein

Forward primer 1 ATGACAGACACGACGTTGCC 20
Template 1402468 GGCG.GCC........C... 1402487

Forward primer 1 ATGACAGACACGACGTTGCC 20
Template 1405472 ..C..G..GGT........G 1405453


product length = 1630
Features associated with this product:
dihydrolipoamide dehydrogenase

hypothetical protein

Forward primer 1 ATGACAGACACGACGTTGCC 20
Template 553845 TG.T.G.C........G... 553864

Forward primer 1 ATGACAGACACGACGTTGCC 20
Template 555474 GCC.GTTC........C... 555455


product length = 4952
Features associated with this product:
nickel-transport integral membrane protein

short chain dehydrogenase

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 3172679 G.G..C...GAC........ 3172660

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 3167728 GCC.......TG........ 3167747


product length = 6002
Features associated with this product:
mannosyltransferase

pterin-4-alpha-carbinolamine dehydratase

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 1292184 CGTCG........GC.G... 1292165

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 1286183 GCCTGG...G.........A 1286202


product length = 893
Features associated with this product:
integral membrane protein

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 682781 .....C.GTGC........A 682762

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 681889 A.C...T.GT........A. 681908


product length = 5280
Features associated with this product:
MerR family transcriptional regulator

integral membrane protein

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 3726125 AGCT..C...........A. 3726106

Reverse primer 1 TTAATTGCCCGTCTGGTCTG 20
Template 3720846 AGGTG..G.T........A. 3720865


Note the different Tm calculation used by the Primer BLAST program... I always anneal at about two degrees under the Primer3 Tm, and it's worked well for me....

HomeBrew on Aug 12 2009, 07:38 PM said:

Greatly appreciated, this has cleared up things a ton for me! I never thought a Red Sox fan would've helped me after last weekend
One more question though, and that's it, I promise, I noticed you have the reverse primer as " TTAATTGCCCGTCTGGTCTG" why wouldn't it be "GTCTGGTCTGCCCGTTAATT" instead? Why is the stop codon in the beginning of that sequence instead of the end of that sequence? That confuses me a little. Other than that, I can just throw on the vector overhang sequence and be OK correct? Thanks again!

it's a common mistake people make when first learning how to design effective primers. as a result, the pcr fails.

remember that the reverse primer 5' end is actually priming from the stop codon. you have to "flip" the primer around in your "mind's eye" so to speak.

5'TTAATTGCCCGTCTGGTCTG3' will touch down in this orientation 3'GTCTGGTCTGCCCGTTAATT5'

which looks like this:

5'CAGACCAGACGGGCAATTAA3'
3'GTCTGGTCTGCCCGTTAATT5'

eldon on Aug 13 2009, 10:53 AM said:

Ah, ok, that makes sense. So when I put in my vector over hangs, I'd actually put them in the beginning of the primer then. So for my two primers I have this:

Forward: GACGACGACAAGATGACAGACACGACGTTGCC
Reverse: GGCCCGAAGAGGAGTTAATTGCCCGTCTGGTCTG

Bolded parts are vector overhangs.

I BLASTED those two primers and it says specified primers are not in sequence, but I'm guessing that's because those overhangs match nothing in the genome, but I can assume these primers should work though? Thanks a ton guys, I would buy yous a beer if I could.

NYYFan on Aug 13 2009, 04:32 PM said:

Correct -- assuming that those are the correct bases for the overhangs you need and that *they* are in the 5' -> 3' orientation.

NYYFan on Aug 13 2009, 04:32 PM said:

Yes. That's why when I BLASTed them here, I left the overhangs off and got:

HomeBrew on Aug 12 2009, 11:38 PM said:

Note that this program expects primers in the 5'->3' orientation also, and that all primer synthesis companies expect the sequences in this orientation as well.

NYYFan on Aug 13 2009, 11:59 AM said:

It was a tough weekend for sure, but some things are (at least marginally) more important than others. At least we took the last 8...

HomeBrew on Aug 12 2009, 09:48 AM said:

NYYFan on Aug 12 2009, 10:12 AM said:

Hi,
To screen for primers, I prefer using NetPrimer available at: http://www.premierbiosoft.com/netprimer/index.html
It provides reliable results, moreover its quite handy.

Cheers