Download Gene Cloning

GENE CLONING TOOLS
Genetic engineering
Gene Cloning allows the separation and identification of a
specific section of genetic material (DNA or RNA) from other
sequences. It then allows the isolation of large numbers of
copies of this sequence for molecular characterisation
Other terms that you will see that mean the same thing include:
DNA cloning
molecular cloning
recombinant DNA technology
What is a gene and what is a coding region?
A gene is a nucleic acid sequence that code for a polypeptide or chain that has a
function in an organism
A gene sequence includes regulatory regions that are responsible for controlling
the spatial and temporal expression of the gene product (a protein or RNA)
A protein is encoded by a coding region which is the part of the gene between the
translation initiation codon (normally ATG)and the translation termination codon
(TAA, TGA or TAG)
It is important that you appreciate the difference between a gene and a coding
region.
In many genetic engineering experiments we will wish to express a protein and so
will only be interested in the coding region, not in the remainder of the gene from
which it is derived.
Some uses of genetic engineering
Cloning allows full characterisation of a gene including identification and
analysis of regulatory sequences and mechanisms controlling spatial and
temporal gene expression (i.e. when and where the gene is expressed) by;
DNA sequence analysis
Determination of 5' and 3' ends of the mRNA transcript
Location of introns/exons,
Analysis of mutated forms of DNA
Transcription control elements
Trans-factors
Sequences that control transcript stability
Localisation of expressed protein
Reverse genetics
Some uses of genetic engineering
2. Genome mapping and evolutionary studies
3. Expression of recombinant proteins, from the coding region, for
structural and functional studies or large scale production of
industrial or medical proteins
3. Protein engineering and directed evolution to generate new
functional proteins
4. Diagnosis of human genetic diseases/ Forensic analysis
5. Gene therapy
6. Transgenic plants and animals
Tools and techniques
We use a range of enzymes as basic tools
to manipulate DNA and RNA during gene
cloning and analysis processes
Restriction enzymes (site-specific cutting)
Phosphatases (removing 5’ phosphates)
Kinases (adding 5’ phosphates)
Ligases (joining fragments)
Nucleases (removing DNA)
Oligonucleotides (synthetic DNA eg primers and probes)
DNA polymerases (replicating, amplifying)
sequencing, PCR, mutagenesis
eg. DNA
Tools and techniques
We use various approaches to investigate genes, gene expression
and to characterise where and when a gene is expressed, and
where and when its product is localised and active.
Gene Cloning: Vectors, enzymes, PCR, agarose gels
Genes and polymorphisms: Southern blot, DNA
sequencing, Next generation sequencing
Transcript analysis: Northern blot, intron/exon, start site
mapping, in situ hybridisation
Global analysis: microarrays, proteomics, transgenic
knockout/in, Next generation sequencing
Protein expression profiles: western blot,
immunocytochemistry, GFP, fusion proteins
Protein expression studies: over-expression, functional analysis
in cells
Molecular interactions: immunoprecipitation, phage display,
yeast hybrid systems, FRET, SPR,
Experimental design sub-cloning
Non-coding regions
Catalase coding region
?
How can I sub-clone this catalase coding region into an
protein expression vector so that I can express and purify the
catalase?
What steps would I need to follow and what tools/techniques
would I need to use?
Let’s think about what tools are available.
Key molecular biology tools
1.
2.
3.
4.
5.
6.
7.
8.
Vectors
Agarose gels
Restriction enzymes: cut DNA
Modifying enzymes: remove or add chemical group (eg
phosphate or nucleotide)
Ligases: join DNA
Polymerases: synthesise DNA (& RNA) and/or remove
nucleotides
Synthetic DNA – oligonucleotides, synthetic genes
Polymerase chain reaction PCR
Now let’s consider a basic gene cloning flow diagram
Basic Steps in Cloning
Purify vector DNA (e.g.
plasmid or phage)
Alkaline lysis
Purify target DNA to
be cloned eg
genomic, cDNA, or in
silico sourced clone
Digest the circular
plasmid DNA with
Restriction enzyme(s)
Alakaline phosphatase
treat the plasmid DNA
to remove 5’ P’s
Screen colonies to identify
those with recombinant
plasmid
Colony PCR or plasmid
isolation & restriction digest
Digest the target DNA
to be cloned with
PCR amplify a DNA
Restriction enzyme(s)
fragment with
carefully designed
primers & digest
Ligate (join) digested
vector and target DNA
Mixture of vector &
recombinants
Colonies form on
plates by cell growth &
plasmid replication to
give a clone
Transform ligation mix
into competent E. coli.
One cell takes up one
DNA molecule
Plate onto agar with
antibiotic
Only plasmid
containing cells grow
First I need to prepare DNA for cloning
PLASMID
DNA
cDNA
/oligo dT
purification of
mRNA
http://www.acgtinc.com/
http://www.acgtinc.com/
GENOMIC
DNA
http://www.pharmatech.co.kr/
First you need to prepare DNA for cloning
cDNA
/oligo dT purification
of mRNA
http://www.acgtinc.com/
We are going to recover the catalase coding region from cDNA that is synthesised
from mRNA.
The mRNA must be isolated from the correct cells and purified (ca. 3-5%) from other
RNAs
This is done by using an oligo dT column or oligo dT magnetic beads to isolated
mRNA which is polyadenylated.
cDNA synthesis then relies upon the enzyme Reverse transcriptase and a primer,
usually an oligo dT primer for first strand synthesis and then a self-priming or
specific primer plus a DNA polymerase for second strand synthesis.
If we know the gene sequences we can actually design two primers that are specific
for the coding region for use in first strand cDNA synthesis followed by PCR
Let’s assume that we are starting with a collection of oligodTprimed cDNA molecules; some of these will be ones that
contain our catalase sequence
Catalase coding region
We know the sequence of the gene from genome sequencing projects and can
access this information from databases such as Genbank
So we can design primers that can be used for PCR amplification of only the
coding region of the cDNA
Polymerase Chain Reaction
http://www.youtube.com/watch?v=eEcy9k_KsDI
PCR involves thermal cycling – 25-40 cycles
Initial
Denaturation
Cycle 1
Cycle 2
94 oC
Te m p e r a t u r e
Thermostable DNA
polymerases:
Denaturation
etc
DNA synthesis at high
temperatures in PCR and
other reactions
Taq
• 5’ to 3’ exonuclease
and 5’ to 3’ DNA
synthesis
72 oC
o
55 C
Annealing
Extension
Time
Kod, Pfu
• 5’ to 3’ DNA synthesis
and 3’ to 5’
exonuclease (proofreading)
Things to consider in PCR primer design
•
About 20 nt long primers
• ~50 % GC if possible and with similar TM >55 oC
•
Avoid complementary primer sequences
•
Avoid polypyrimidine (T, C) or polypurine (A, G) stretches
• Can add sequences to 5’-end
Eg. Restriction enzyme site
Promoter sequence eg T7 DNA pol
can be added to the 5’ end
5'
primer
3'
tem plate
How do we design primers for PCR?
We know the sequence of the gene from genome sequencing projects and can
access this information from databases such as Genbank
So we can design primers that can be used for PCR amplification of only the
coding region of the cDNA
ATG
5’
TAA
TGA
TAG
3’
3’
5’
But….how will we be able to clone this PCR amplified coding sequence into a
cloning vector?
First we need to decide what cloning vector we will use
Common cloning vectors
Plasmids
Viruses/Bacteriophage
Cosmids
• combination of plasmid and bacteriophage l
Phagemids
– combination of plasmid and bacteriophage M13
T7 TERM
pET28 plasmid
vector
His6 tag
An example of a
cloning vector used
routinely in my lab
XhoI (5207)
NotI (5199)
HindIII (5192)
SalI (5186)
SacI (5183)
EcoRI (5173)
BamHI (5167)
Multiple cloning site
expression region
NheI (5135)
TEV cleavage site
Purification of
expressed protein
f1 ori
KpnI (5112)
Antibiotic resistance
kanamycin
resistance
Kanr
His6 tag
NcoI (5070)
XbaI (5031)
Promoter
T7 P
BglII (4965)
SphI (4776)
Expressed gene regulation
lacI
XmaI (1068)
SmaI (1070)
pET28 TEV
ClaI (1251)
5368 bp
EcoRV (3797)
Origin of replication
pBR322 ori
Designing the primers for PCR?
If we are going to clone into pET28 we will need to add restriction sites at the
ends of the coding region
We do this by adding the restriction enzyme cleavage sequences to the 5’ end
of the primers.
If we add different sites to the 5’ and 3’ end of the coding region then we can do
directional cloning so that we know the sequence is inserted into the vector in
the correct orientation.
We will add an NcoI site at the 5’ end of coding region and EcoRI site at the 3’ end
3’
5’
3’
5’
When we PCR amplify the coding region using these primers we will
generate this sequence
EcoRI
We need to check whether we have the correct PCR product and digested vector
A more detailed look at how to design the primers for
PCR of the coding region
Always label 5’ and 3’ ENDS when writing
5’
3’
CATCTGCTAGTCCAACCTACATCATGTCGTCAAGTCAT-1kb-ATTATTATCTCTCTGGATGTCAACATGAAACACCTGCTAACACTC
3’
GTAGACGATCAGGTTGGATGTAGTACAGCAGTTCAGTA-1kb-TAATAATAGAGAGACCTACAGTTGTACTTTGTGGACGATTGTGAG
5’
Select primer sites (ca. 20 nt)
5’
3’
CATCTGCTAGTCCAACCTACATCATGTCGTCAAGTCAT-1kb-ATTATTATCTCTCTGGATGTCAACATGACACACCTGCTAACACTC
GTAGACGATCAGGTTGGATGTAGTACAGCAGTTCAGTA-1kb-TAATAATAGAGAGACCTACAGTTGTACTGTGTGGACGATTGTGAG
3’
5’
Add sequences onto primer with a few extra 5’ nucleotides to ensure efficient
restriction enzyme cleavage.
NcoI = CCATGG; EcoRI = GAATTC
5’
3’
CATCTGCTAGTCCAACCTACATCATGTCGTCAAGTCAT-1kb-ATTATTATCTCTCTGGATGTCAACATGAAACACCTGCTAACACTC
GTAGACGATCAGGTTGGATGTAGTACAGCAGTTCAGTA-1kb-TAATAATAGAGAGACCTACAGTTGTACTTTGTGGACGATTGTGAG
5’ ATCC CCATGG GTCCAACCTACATCATGTCG 3’
3’ GACCTACAGTTGTACTTTGT GAATTCTGCT 5’
4 nts
EcoRI
NcoI
Always write primers as 5’ to 3’
sequences so the reverse strand needs
rewritten
5’ TCGTCTTAAGTGTTTCATGTTGACATCCAG 3’
PCR
NcoI
4 nts
EcoRI
3’
5’
Analysing DNA fragments by agarose gel electrophoresis
• Electrophoresis through an agarose gel matrix
• At neutral pH DNA and RNA have a net NEGATIVE
charge due to phosphate groups and so move
towards the ANODE (+ve electrode)
• Small molecules move through faster than
longer/larger molecules so separation is on the
basis of size
– For linear fragments rate of migration
proportional to log10 molecular size
Can also separate on basis of conformation
Plasmid DNA: Supercoiled, open circular
and linear are all the same molecular size
but migrate differently
T7 TERM
His6 tag
Next we need to restriction
digest the vector and PCR
product with NcoI and EcoRI
XhoI (5207)
NotI (5199)
HindIII (5192)
SalI (5186)
SacI (5183)
EcoRI (5173)
BamHI (5167)
expression region
NheI (5135)
TEV cleavage site
f1 ori
KpnI (5112)
His6 tag
NcoI (5070)
Kanr
XbaI (5031)
T7 P
BglII (4965)
SphI (4776)
lacI
XmaI (1068)
SmaI (1070)
pET28 TEV
ClaI (1251)
5368 bp
EcoRV (3797)
pBR322 ori
Restriction enzymes
Endonucleases: Digest DNA at internal (often palindromic)
sites in DNA
– Restriction enzymes cleave DNA only at specific
recognition sites
– generating fragments for cloning
– map genes and polymorphisms (SNP’s)
5’
3’
5’
3’
5’
3’
GAATTC
CTTAAG
3’
GAATTC
CTTAAG
3’
G3’
CTTAA5’
5’AATTC
3’G
5’
5’
3’
5’
Animation: Restriction enzymes
• http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites
/dl/free/0072437316/120078/bio37.swf::Restriction
Endonucleases
Restriction enzyme sites
• Restriction enzymes can leave three different types of ends
– 5’ overhang (sticky end)
– 3’ overhang (sticky end)
– blunt
Eco RI
GAATTC
CTTAAG
G3’
CTTAA5’
5’AATTC
5’ OVERHANG
3’G
Pvu II
Kpn I
CAGCTG
GTCGAC
GGTACC
CCATGG
CAG3’
GTC5’
5’CTG
3’GAC
BLUNT END
GGTAC3’
C5’
5’C
3’CATGG
3’ OVERHANG
The ends generated allow different DNA fragments to be joined
Restriction enzyme sites
• Some enzymes recognise different sites but generate the
SAME sticky ends
Bam HI
Bgl II
GGATCC
CCTAGG
AGATCT
TCTAGA
Bam HI end
G3’
CCTAG5’
Bgl II end
5’GATCT
+
3’A
Sau 3A
NGATCN
NCTAGN
Product
GGATCT
CCTAGA
Will not cut with Bam HI
or Bgl II, but will still cut
with Sau 3A
Often the restriction digested vector DNA is also treated with the
enzyme
• Alkaline phosphatase:
– removes the 5’ phosphate groups from DNA, normally
the vector DNA
– needs inactivated usually by heat before the ligation
step (otherwise it can dephosphorylate the insert as
well!!)
Hydrolysis of phosphate ester
-3
+ 2 PO 4
Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A. Brown.
Why do we use alkaline phosphatase?
Vector
Vector plus insert
3’OH
5’PGATC
CATG5’
3’OH
P
GATC
CATG
Vector
Vector with NO insert
3’OH
Ligation
5’OH
CATG5’OH
Insert
GATC
Vector
3’OH
GATC
CATG
No ligation
The vector and insert DNA are then mixed in a ligase buffer
containing ATP and DNA ligase
• Generates phosphodiester bonds between 3’OH and 5’ P
• Must have a 5’Phosphate (5’P) for ligase to function
Vector
Join two double strand
molecules together if they
have suitable ends
3’OH
5’PGATC
CATGP5’
3’OH
GATC
CATG
3’OH 5’P
Repair single-strand breaks
in the phosphodiester
backbone
useful in some site-directed
mutagenesis applications
O-P-O
Insert
Following a ligation reaction an aliquot is
transformed into competent E. coli cells.
The E. coli cells are treated with CaCl2 or
RbCl2 to disrupt their cell walls and can
be stored frozen at -80oC.
For a transformation reaction aliquots of
cells are thawed on ice and DNA is
added, typically around 40 -5 0 ng.
After incubating on ice the cells are heat
shocked for around 1-2 min at 42oC so
that cells take up the DNA
Very few of the cells will actually become
transformed and so we need to be able to
identify those cells that have been
transformed and we do this by antibiotic
selection
Selection and screening
All the transformed
colonies will contain a
vactor, but NOT all will
contain recombinant
plasmids
(1) Clones containing vector molecules can grow –
they are antibiotic resistant!
• Reduce vector only (eg alkaline
phosphatase)
(2) How do you identify recombinants?
Blue white selection (based on lacZ activity)
Colony PCR
Purify plasmid & restriction digest
Hybridization screening
Most common
then
DNA sequence
DNA Polymerases
Uses: DNA synthesis (and sometimes as an
exonuclease) DNA sequencing DNA mutagenesis
DNA labelling
E. coli DNA polymerase I: synthesises DNA using a
template and primer
– Three activities:
•
•
•
5’-3’ exonuclease (repair)
5’-3’ DNA synthesis
3’-5’ exonuclease (proof-reading)
Klenow fragment
T4 DNA pol
T7 DNA pol
These are useful for some DNA manipulation including
• Filling in sticky ends to make them blunt ends
• Radioactive labelling ends
• DNA synthesis reactions that use a primer
DNA Polymerases that are now more
commonly used than Pol I
•
T7 and T4 phage DNA polymerases:
–
•
Klenow activities, but more efficient
Thermostable DNA polymerases:
–
DNA synthesis at high temperatures in PCR and
other reactions
Taq
–
•
–
Kod, Pfu
•
•
5’ to 3’ flap exonuclease and 5’ to 3’ DNA synthesis
5’ to 3’ DNA synthesis and 3’ to 5’ exonuclease (proofreading)
Reverse transcriptase:
–
synthesises cDNA using RNA as a template and a
DNA primer
Reading associated with this lecture
1. Gene cloning essentials
1.1 Introduction
1.2 Gene cloning applications
1.3 Gene cloning in the laboratory
1.4 Gene cloning processes
1.5 Further types of gene cloning
1.6 Chapter summary
3
4
4
5
14
18
21
2. Polymerase chain reaction
23
2.1 Introduction
23
2.2 How PCR works
24
2.3 The PCR protocol
26
2.4 PCR techniques and applications 31
2.5 Forensic DNA analysis
40
2.6 Future prospects
41
2.7 Chapter summary
41