plasmids, primers (and beyond!). or how to make green mice…

Plasmids, primers (and beyond!)

Or how to make green mice….

Foreign DNA can be incorporated into a cloning vector (plasmid, phage, YAC or other) if both foreign DNA and vector are cleaved with the same restriction endonuclease. The pieces of DNA will anneal, and then can be ligated using DNA ligases. The desired fragment can be separated from others using gel electrophoresis.

Cleavage of DNA by restriction endonucleases, followed by incorporation of “foreign” DNA into a a plasmid using restriction endonucleases. Insertion is facilitated by the annealing of “sticky” ends of the restriction fragments. (Some restriction enzymes give blunt ends: annealing then requires complementary blunts ends. In either case, ligation is done by a DNA ligase.

Restriction enzymes: Sticky ends vs. Nonsticky ends

Adding restriction sites

• We can design primers to add specific sequences of DNA to the beginning and end of our insert DNA.

• When we want to ligate a piece of DNA(in our case mefp-5) into a plasmid we use restriction enzymes. Restriction enzymes recognize and cleave certain palindromic pieces of DNA.

Synthetic DNA with several cleavage sites can be ligated into a vector to create a synthetic polylinker site in the vector.

Cohesive ends can be formed on a DNA fragment (e.g., a synthetic oligonucleotide) by adding a short segment using a DNA ligase (such as the one from T4 ligase). The short segment contains a cleavage site for a restriction enzyme, in this case, EcoR1. EcoR1 forms an overhanging adhesive site.

Tags?Tags are added to your insert DNA in order to purify the resultant protein after expression. For instance, a his-tag, a sequence of 6 histidines, will cause the protein to stick to certain kinds of chromatography columns. After washing out all other proteins in your cell lysate with buffer only your protein of interest will remain. This protein can be removed from the column with certain harsher chemicals.

Adding tags• Always make sure your

insert is IN FRAME!• For N-terminal tags, make

sure to add a stop codon if there isn’t one already.

• Alternatively, for C-terminal tags, make sure your insert doesn’t have a stop codon.

• ON THE MAP:• From left to right the order

of transcription is N-terminus to C-terminus

GAATTCGCGGCCGCTTCTAGGCATGAAGCTGTCGTGCATCGTCCTGGTCCTCTTCCTGGTCACGCTGGCGGCTTACTCCGACGTGGGCTCGTCCTCGTCGGAGGAGTATAAGGGCGGCTATTACCCAGGCAATGCCTATCACTATCATAGTGGAGGCTCTTACCACGGCAGCGGCTATCACGGTGGCTACAAGGGCAAGTACTACGGTAAGGCGAAGAAATATTACTATAAGTACAAGAACTCGGGCAAGTACAAATATCTCAAGAAGGCCCGCAAATACCATCGCAAGGGCTATAAGTACTACGGCGGCTCGTCGTACTAGTAGCGGCGGCTCCAGAGATCT

Outline of a primer:• Forward primer:

– Junk DNA+Restriction site+Junk DNA to keep in frame+12 bp of sequence

• Reverse primer:– Take reverse complement of (Junk DNA+12bp of

sequence+Junk DNA to keep in frame+restriction site)

• Why junk DNA at the beginning of your primer?– Necessary in order for transcription machinery to

bind to your sequence.

pET 28a!

A closer look• Enzymes:

– EcoRI: G|AAT TC– NotI:GC|GGCCGC– NcoI: C|CATGG– NdeI: CA|TATG

N-terminal tagThe beginning of mefp-5 is (in frame) GCA TGA AGC TGT CGT

WE MUST PRESERVE THIS PATTERN.

•If we put an EcoRI site on the N-terminus of our mefp-5 insert DNA, our forward primer will be

•G|AATTC GCATGAAGCTGTCGT•…as will the first

•When we cut mefp-5 the result is (in frame)•AAT TCG CAT GAA GCT GTC GT

•…..WHICH IS OUT OF FRAME

To fix this, add an extra piece of “junk” DNA to shift the reading frame back in its proper place

•G|AATTC A GCATGAAGCTGTCGTAAT TCA GCATGAAGCTGTCGT

Hooray! Back in our proper reading frame!

N-terminal tag…continuedSo what if we ONLY want an N-terminal tag,

but NO C-terminal tag• One word: Stopcodons*

• Insert a TAA at the N-terminal end of your sequence BEFORE the restriction site– Plasmids like pET28a contain a stop codon AFTER the C-terminal histidine tag. This

signals transcription to stop after the C-terminal histidine tag has been transcribed.– We want transcription to stop BEFORE the C-term tag. So if we use NotI as our C-

terminal restriction enzyme for our mefp-5 insert, the end of the sequence should look like (NotI is GC|GGCCGC)

TAC TAC GGC GGC TCG TCG TAA GC|GGCCGCWhich corresponds to the reverse primer

GCGGCCGC TTA CGACGAGCCGCCGTAGTA

*of course it’s a real word.

Adding a C-term tag• His-tag must be in frame. But how?

– Use different enzymes– Add extra bp

EcoRI-Reverse: TACTACGGCGGCTCGTCG T G|AATTCPrimer: GAATTC CGACGAGCCGCCGTAGTA *orange=junk DNA to keep in frame

NotIReverse: TAC TAC GGC GGC TCG TCG GC|GGCCGCPrimer: GCGGCCGC CGACGAGCCGCCGTAGTA

pGEX!

•Straightforward N-terminal GST tag•Keep in frame!

•Two variants we have, 4T-1 and 4T-3 just have different reading frames•Stop codon is included at C-terminus•Thrombin cleavage to cleave GST tag from your purified protein