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:
GAATTCGCGGCCGCTTCTAGGCATGAAGC
TGTCGTGCATCGTCCTGGTCCTCTTCCTGG
TCACGCTGGCGGCTTACTCCGACGTGGGC
TCGTCCTCGTCGGAGGAGTATAAGGGCGG
CTATTACCCAGGCAATGCCTATCACTATCAT
AGTGGAGGCTCTTACCACGGCAGCGGCTA
TCACGGTGGCTACAAGGGCAAGTACTACG
GTAAGGCGAAGAAATATTACTATAAGTACA
AGAACTCGGGCAAGTACAAATATCTCAAG
AAGGCCCGCAAATACCATCGCAAGGGCTA
• From left to right the order TAAGTACTACGGCGGCTCGTCGTACTAGTA
of transcription is NGCGGCGGCTCCAGAGATCT
terminus to C-terminus
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 tag
The 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 GCATGAAGCTGTCGT
AAT TCA GCATGAAGCTGTCGT
Hooray! Back in our proper reading frame!
N-terminal tag…continued
So 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 Cterminal 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|GGCCGC
Which 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
NotI
Reverse: TAC TAC GGC GGC TCG TCG GC|GGCCGC
Primer: GCGGCCGC CGACGAGCCGCCGTAGTA
EcoRI
-Reverse: TACTACGGCGGCTCGTCG T G|AATTC
Primer: GAATTC CGACGAGCCGCCGTAGTA
*orange=junk DNA to keep in frame
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