1. GENE CLONING TOOLS

2. 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

3. 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.

4. 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

5. 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
Protein engineering and directed evolution to generate new functional proteins
Diagnosis of human genetic diseases/ Forensic analysis
Gene therapy
Transgenic plants and animals

6. 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) eg. DNA sequencing, PCR, mutagenesis

7. 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,

8. Catalase coding region
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.

9. Key molecular biology tools
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

10. Basic Steps in Cloning
Purify target DNA to be cloned eg genomic, cDNA, or in silico sourced clone
Purify vector DNA (e.g. plasmid or phage)
Alkaline lysis
Digest the circular plasmid DNA with
Restriction enzyme(s)
Digest the target DNA to be cloned with
Restriction enzyme(s)
PCR amplify a DNA fragment with carefully designed primers & digest
Alakaline phosphatase treat the plasmid DNA
to remove 5’ P’s
Ligate (join) digested vector and target DNA
Mixture of vector & recombinants
Transform ligation mix into competent E. coli. One cell takes up one DNA molecule
Plate onto agar with antibiotic
Only plasmid containing cells grow
Colonies form on plates by cell growth & plasmid replication to give a clone
Screen colonies to identify those with recombinant plasmid
Colony PCR or plasmid isolation & restriction digest

11. First I need to prepare DNA for cloning
cDNA
PLASMID
DNA
/oligo dT purification of mRNA
GENOMIC
DNA

12. First you need to prepare DNA for cloning
/oligo dT purification of mRNA
cDNA
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

13. Catalase coding region
Let’s assume that we are starting with a collection of oligodT-primed 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

14. Polymerase Chain Reaction

15. PCR involves thermal cycling – 25-40 cycles
Thermostable DNA polymerases:
DNA synthesis at high temperatures in PCR and other reactions
Taq
5’ to 3’ exonuclease and 5’ to 3’ DNA synthesis
Kod, Pfu
5’ to 3’ DNA synthesis and 3’ to 5’ exonuclease (proof-reading)
72 C o
94 C
55 C
etc
Initial
Denaturation
Cycle 1
Cycle 2
Time T e m p r a t u
Annealing
Extension

18. 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

19. 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 5’ 3’
ATG
TAA
TGA
TAG
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

20. Common cloning vectors
Plasmids
Viruses/Bacteriophage
Cosmids
combination of plasmid and bacteriophage l
Phagemids
combination of plasmid and bacteriophage M13

21. Antibiotic resistance kanamycin resistance
pET28 plasmid vector
Purification of expressed protein
An example of a cloning vector used routinely in my lab
Multiple cloning site
Antibiotic resistance kanamycin resistance
Promoter
Expressed gene regulation
Origin of replication

22. 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. 5’ 3’
We will add an NcoI site at the 5’ end of coding region and EcoRI site at the 3’ end
NcoI
EcoRI
EcoRI
NcoI
When we PCR amplify the coding region using these primers we will generate this sequence
We need to check whether we have the correct PCR product and digested vector

23. A more detailed look at how to design the primers for PCR of the coding region
NcoI
EcoRI
CATCTGCTAGTCCAACCTACATCATGTCGTCAAGTCAT-1kb-ATTATTATCTCTCTGGATGTCAACATGAAACACCTGCTAACACTC
GTAGACGATCAGGTTGGATGTAGTACAGCAGTTCAGTA-1kb-TAATAATAGAGAGACCTACAGTTGTACTTTGTGGACGATTGTGAG 5’ 3’
Always label 5’ and 3’ ENDS when writing
Select primer sites (ca. 20 nt)
CATCTGCTAGTCCAACCTACATCATGTCGTCAAGTCAT-1kb-ATTATTATCTCTCTGGATGTCAACATGACACACCTGCTAACACTC
GTAGACGATCAGGTTGGATGTAGTACAGCAGTTCAGTA-1kb-TAATAATAGAGAGACCTACAGTTGTACTGTGTGGACGATTGTGAG 5’ 3’

24. NcoI = CCATGG; EcoRI = GAATTC
CATCTGCTAGTCCAACCTACATCATGTCGTCAAGTCAT-1kb-ATTATTATCTCTCTGGATGTCAACATGAAACACCTGCTAACACTC
GTAGACGATCAGGTTGGATGTAGTACAGCAGTTCAGTA-1kb-TAATAATAGAGAGACCTACAGTTGTACTTTGTGGACGATTGTGAG 5’ 3’
Add sequences onto primer with a few extra 5’ nucleotides to ensure efficient restriction enzyme cleavage.
NcoI = CCATGG; EcoRI = GAATTC 3’
GTCCAACCTACATCATGTCG 3’
GACCTACAGTTGTACTTTGT 5’
4 nts
ATCC
NcoI
CCATGG
EcoRI
GAATTC 5’
4 nts
TGCT
TCGTCTTAAGTGTTTCATGTTGACATCCAG 5’ 3’
Always write primers as 5’ to 3’ sequences so the reverse strand needs rewritten
NcoI EcoRI
PCR

25. 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

26. Next we need to restriction digest the vector and PCR product with NcoI and EcoRI

27. 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’
GAATTC
CTTAAG 3’ 3’ 5’ 5’ 3’
GAATTC
CTTAAG 5’ 3’
G3’
CTTAA5’
5’AATTC
3’G

28. Animation: Restriction enzymes
Endonucleases

29. Restriction enzyme sites
Restriction enzymes can leave three different types of ends
5’ overhang (sticky end)
3’ overhang (sticky end)
blunt
Eco RI Pvu II Kpn I
GGTACC
CCATGG
GAATTC
CTTAAG
CAGCTG
GTCGAC
G3’ ’AATTC
CTTAA5’ ’G
CAG3’ 5’CTG
GTC5’ 3’GAC
GGTAC3’ ’C
C5’ ’CATGG
5’ OVERHANG BLUNT END ’ OVERHANG
The ends generated allow different DNA fragments to be joined

30. Restriction enzyme sites
Some enzymes recognise different sites but generate the SAME sticky ends
Bam HI Bgl II Sau 3A
GGATCC
CCTAGG
AGATCT
TCTAGA
NGATCN
NCTAGN
G3’
CCTAG5’
5’GATCT
3’A +
GGATCT
CCTAGA
Will not cut with Bam HI or Bgl II, but will still cut with Sau 3A
Bam HI end Bgl II end Product

31. Alkaline phosphatase:
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.

32. Why do we use alkaline phosphatase?
3’OH ’PGATC
CATG5’ ’OH
GATC
CATG
Vector
Insert
Ligation
Vector plus insert P
GATC
CATG
No ligation
3’OH ’OH GATC
CATG5’OH ’OH
Vector
Vector with NO insert

33. Generates phosphodiester bonds between 3’OH and 5’ P
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
3’OH ’PGATC
CATGP5’ ’OH
GATC
CATG
Vector
Insert
Join two double strand molecules together if they have suitable ends
3’OH 5’P
O-P-O
Repair single-strand breaks in the phosphodiester backbone
useful in some site-directed mutagenesis applications

34. 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 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

35. Selection and screening
All the transformed colonies will contain a vactor, but NOT all will contain recombinant plasmids
Clones containing vector molecules can grow – they are antibiotic resistant!
Reduce vector only (eg alkaline phosphatase)
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

36. 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)
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
Klenow fragment
T4 DNA pol
T7 DNA pol

37. 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
5’ to 3’ flap exonuclease and 5’ to 3’ DNA synthesis
Kod, Pfu
5’ to 3’ DNA synthesis and 3’ to 5’ exonuclease (proof-reading)
Reverse transcriptase:
synthesises cDNA using RNA as a template and a DNA primer

38. Reading associated with this lecture
1. Gene cloning essentials
1.1 Introduction
1.2 Gene cloning applications
1.3 Gene cloning in the laboratory 5
1.4 Gene cloning processes
1.5 Further types of gene cloning
1.6 Chapter summary
2. Polymerase chain reaction
2.1 Introduction
2.2 How PCR works
2.3 The PCR protocol
2.4 PCR techniques and applications 31
2.5 Forensic DNA analysis
2.6 Future prospects
2.7 Chapter summary