2. Primer Design
2.1 Multiple Cloning Sites
All commonly-used expression vectors used in the Jia Lab contain the following multiple cloning site: BamHI EcoRI SmaI SalI XhoI NotI
XXX XXX GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC XXX XXX
XXX XXX CCT AGG GGC TTA AGG GCC CAG CTG AGC TCG CCG GCG XXX XXX
XXX XXX Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg XXX XXX
When designing your primers, you should add one of these restriction sites to the end of your primer in such as was as to preserve the reading frame shown here at both the 5’ and the 3’ end of the gene!!!. This is really, really important for insuring that you express a properly tagged protein.
Here are the actual multiple cloning sites of our expression plasmids, including tags, stop codons, and other ‘business’:
P1 (pGEX-4T-3):
BamHI EcoRI SmaI SalI XhoI NotI Stop Codons
CTGGTTCCGCGT GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA
GACCAAGGCGCA CCT AGG GGC TTA AGG GCC CAG CTG AGC TCG CCG GCG TAG CAC TGA CTG ACT
LeuValProArg*Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp ***
P13 (pET16bMCS):
ATG GGC CATCATCATCATCATCATCATCATCATCAC AGC AGC GGC CAT ATC GAA GGT CGT CAT
水产之书TAC CCG GTAGTAGTAGTAGTAGTAGTAGTAGTAGTG TCG TCG CCG GTA TAG CTT CCA GCA GTA
MET Gly HisHisHisHisHisHisHisHisHisHis Ser Ser Gly His Ile Glu Gly Arg*His
10xHis-Tag Factor Xa
BamHI EcoRI SmaI SalI XhoI NotI
青海大学农牧学院>太阳方位角ATA GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA TAT CCT AGG GGC TTA AGG GCC CAG CTG AGC TCG CCG GCG TAG CAC TGA CTG ACT情报科学 Ile Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp ***
P22 (pET21bMCS):
BamHI EcoRI SmaI SalI XhoI NotI
CATATA GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC GTC GAGCACCACCACCACCACCACTGA
GTATAT CCT AGG GGC TTA AGG GCC CAG CTG AGC TCG CCG GCG CAG CTCGTGGTGGTGGTGGTGGTGACT
Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Val GluHisHisHisHisHisHis***
6xHis-Tag
P259 (pGEXHis):
BamHI EcoRI.SmaI.SalI.XhoI.NotI NdeI
CTGGTTCCGCGT GGATCCCCGAATTCCCGGGTCGACTCGAGCGGCCGCATCGAAGGTCGT CATATGCACCACCACCACCACCACTGA GACCAAGGCGCA CCTAGGGGCTTAAGGGCCCAGCTGAGCTCGCCGGCGTAGCTTCCAGCA GTATACGTGGTGGTGGTGGTGGTGACT
LeuValProArg*GlySerProAsnSerArgValAspSerSerGlyArgIleGluGlyArg*HisMETHisHisHisHisHisHis***
P270 (pPIC6L4zeo):
BamHI EcoRI SmaI SalI XhoI_ NotI
AGC CCT AGG GGC TTA AGG GCC CAG CTG AGC TCG CCG GCG GAG CTG GTAGTAGTAGTAGTAGTAACT
Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Leu Asp HisHisHisHisHisHis***
2.2 Primer Choice Considerations
•Select the first (or last) 21 bases of your gene of interest as the basis of your primer. This is the complementary region that will be used to perform PCR to amplify your gene of interest.
Keep in mind that DNA is synthesized 5’Æ3’, and so you’re 5’ forward primer should be
complementary to the bottom strand of you gene of interest, and the 3’ reverse primer should be complementary to the top strand of your gene of interest. See the example at the end of this
section.
•The 5’ (forward) primer must contain a start codon (ATG), and if you think you might want to express the protein-of-interest in yeast (Pichia pastoris), make sure this start codon is part of
a Pichia Kozak consensus (which is (G/A)NN ATG G).
•Consider the amino acids you will be adding to the protein of interest with this primer. In order to shift the reading frame appropriately, you will often have to add a few bases to the
restriction sites in your primer. Also, adding the Kozak consensus above will often require
You should figure out which amino acids these extra sequences will encode in the protein-of-
interest—if possible avoid introducing ‘weird’ amino acids with large functional groups
(tryptophan, phenylalanine, etc) or unique chemical/structural properties (cysteine). I usually look at the multiple cloning site and pick an amino acid residue encoded within it as a guide to relatively structurally neutral amino acids.
•Avoid chosing SmaI as a restriction site for cloning. The Pichia expression vector (p270, or pPIC6L4zeo) contains two SmaI sites (one within the multiple cloning site, plus another within the Zeocin resistance cassette). For you, this means that SmaI can not be used to clone your
gene of interest in to the Pichia vector.
•Choose a restriction site that is not present in the gene you are cloning. For obvious reasons….okay, it’s so that you don’t cut up your gene while doing the cloning.
•If possible, choose BamHI (5’ forward primer) and NotI (3’ reverse primer) to include in your primer design
•Make sure your primer choice will lead to the gene of interest being in the correct reading frame after subcloning. I know I wrote this already, but it’s really crucial and easy to screw up.
•Double and triple check the sequence of the primer you choose. Follow the measure-twice-cut-once maxim, because you don’t want to waste weeks and weeks of work on an incorrect or frame-shifted primer.
2.1 Using DNAMan
2.1.1 Finding the Open Reading Frame
We often obtain cDNA clones of our genes, and wish to subclone them via PCR in to expression vectors. These cDNA clones are very often the product of large-scale cDNA library preparations of mRNA, and therefore contain non-coding sequences that were originally present in the mRNA
1.Load DNAMan and open a text file that contains the cDNA sequence of your gene of interst.
Alternative, copy the sequence of your gene of interest from GenBank, and then (in DNAMan) go to Edit/Enter sequence and Paste it in.
2.Highlight the sequence, then go to Sequence/Load Sequence/From Selection. This loads the
selected sequence in to Channel 1 (NOTE: the number down the left side of the DNAMan
screen are individual channels. Each of these can hold separate protein/DNA sequences. Click on the channel of interest to work with the sequence it contains).
3.Go to Sequence/Search For/Open Reading Frame. Accept all defaults and click ‘Okay’.
4.You will be presented with a list of open reading frames, including the location of both start and
stop codons. Evey gene will have many—in general, you will want the one that codes for the longest protein, but there are exceptions to this rule. You’ll undoubtedly have some extra
information about your protein (ie, length, etc) to allow you to pick the correct reading frame.
2.1.2 Restriction Analysis
In order to find the restriction sites present in your gene of interest, use DNAMan as follows:
1.Load DNAMan and open a text file that contains the cDNA sequence of your gene of interst.
Alternative, copy the sequence of your gene of interest from GenBank, and then (in DNAMan) go to Edit/Enter sequence and Paste it in.
2.Highlight the sequence, then go to Sequence/Load Sequence/From Selection. This loads the
selected sequence in to Channel 1 (NOTE: the number down the left side of the DNAMan
screen are individual channels. Each of these can hold separate protein/DNA sequences. Click on the channel of interest to work with the sequence it contains).
3.Go to Restriction/Restriction Analysis. Here, you will be walked through a series of dialogues
asking a variety of question about the source DNA and the restriction site you want to search
for. I usually select only the six restriction sites of the multiple cloning site to avoid a
confusingly large list of sites. This part of the analysis should be self explanatory.
4.In the end, you will be presented with a list of restriction sites present in the sequence, with a
list of non-cutting enzymes at the bottom. Among the non-cutting enzymes, you should find
two that are present in the multiple cloning site that you can use to subclone your gene of
interest.
2.3 Example: BIN1
Here is the ORF sequence for a gene called bin1:
Coding: 5’-ATGGCAGAGATGGGCAGTAAAGGGGTGACG .other stuff. CCCGAGAACTTCACTGAGAGGGTCCCATGA-3’ Complementary: 3’-TACCGTCTCTACCCGTCATTTCCCCACTGC .other stuff. GGGCTCTTGAAGTGACTCTCCCAGGGTACT-5’
Note the start (ATG) and stop (TGA) codons, the coding strand (shown in the 5’ to 3’ orientation) and the complementary non-coding strand (shown in 3’ to 5’ orientation). The first thing I did was pump the sequence through a restriction site analysis using DNAMan, and found that this sequence contains neither BamHI nor NotI. I will therefore choose these two sites to put in my primers for subcloning. To design the forward primer, I select the first 21 bases or the above sequence:
5’- ATG GCA GAG ATG GGC AGT AAA
Luckily, I don’t need to add a start codon, since I’m cloning this gene from the very beginning and it already has one. If you were cloning a gene from the middle someplace, then you would have to ad庄荣昌
d an ATG here. I then add the BamHI site (GGA TCC) to the 5’ end of this sequence: 5’- gga tcc ATG GCA GAG ATG GGC AGT AAA
Note that, to keep this primer straight in my head, I’ve used lowercase letters to designate sequence that I’ve added/changed to the complementary sequence. This primer seems pretty good, but then I remember that I may want to express this protein in Pichia, so I make some additions and changes to give my final product a Kozak consensus (G/A)NNATGG:
5’- gga tcc gtc ATG GCA GAG ATG GGC AGT AAA
This requires the addition of a ‘GTC’ between the BamHI site and the start codon. Sometimes, you aren’t lucky enough to have ‘G’ immediately downstream of the ATG start codon, and so you have to introduce a mutation here to ensure you have that ATGG start sequence. Finally, I double check which amino acid residues I’ll be adding/changes with this primer:
5’- gga tcc gtc ATG GCA GAG ATG GGC AGT AAA
5’- Gly Ser Val
Nothing weird introduced, so this primer is good to go. I’ll do one last thing, though—I like to add a c
ouple of bases to the 5’ end of the primer to preserve my restriction site during PCR and subsequent TA cloning (errors can be introduced right at the end during PCR, and I want to avoid the damage this can do to my restriction sites). So the final forward primer looks like this:
BIN1-F: 5’- gc gga tcc gtc ATG GCA GAG ATG GGC AGT AAA
For the reverse primer, I start in a similar way by selecting the last 21 bases in this sequence. NOTE that I am EXCLUDING the stop codon—this is really, really important. You will also see that I’m picking a sequence that is complementary to the coding strand (which is the opposite of the forward primer), and that I am writing it in the 5’ to 3’ orientation (required when we’re ordering the primer).
5’- TGG GAC CCT CTC AGT GAA GTT
As above, I will add a NotI (GCG GCC GC) restriction site. Notice that when I write out these sequences, I write them in the triplets that correspond to the proper reading frame (from the multiple cloning sites described above).无石棉板
5’- gcg gcc gc TGG GAC CCT CTC AGT GAA GTT
Right away, I see that if I use this as my primer, I will shift the reading frame by one base (between t
he restriction site and the complementary sequence). So I will add a base to shift the reading frame back in to place:
5’- gcg gcc gct TGG GAC CCT CTC AGT GAA GTT
And then I check the amino acids that will be introduced/modified by this primer. (To do this, keep in mind that this primer corresponds to the non-coding strand, so you have to make your brain read the complement of this primer from right-to-left to correctly guess the amino acids that are encoded: 5’- gcg gcc gct TGG GAC CCT CTC AGT GAA GTT
cgc cgg cga acc ctg ........ect. (READ FROM RIGHT TO LEFT)
Arg Gly Ser Pro Val ........ect.
Everything looks good. The final primer (with an added couple of bases to the 5’ end) looks like this: BIN1-R: 5’- ga gcg gcc gct TGG GAC CCT CTC AGT GAA GTT
So now I am ready to double-check these primers and copy/paste these sequences in to a primer order form:
BIN1-F: 5’- gc gga tcc gtc ATG GCA GAG ATG GGC AGT AAA
BIN1-R: 5’- ga gcg gcc gct TGG GAC CCT CTC AGT GAA GTT
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