1. Primer Design

4. Put in your sequence

5. Primer size
Annealing temperature
% GC

6. Your sequence
Left primer
Right primer
Pick primers

8. Product size
Left primer
Right primer

10. Search for RE site
BioEdit

15. Cloning & Expression Vector

16. DEFINITIONS Clone Cloning
A cell, group of cells, or organism that is descended from and genetically identical to a single ancestor.
An organism descended asexually from a single ancestor.
To make multiple identical copies of a DNA sequence.
To create or propagate an organism from a clone cell.

17. Application Cloning can be used to test for genetic diseases
Regenerate nerves or spinal cord tissue
Help in plastic surgery
Clone organs for transplantation
Grow skin grafts for burn victims
Manufacture bone, fat, and cartilage

18. What is cloning? Reproductive cloning Therapeutic cloning -
9/20/2018 -
What is cloning?
The entire animal is produced from a single cell by asexual reproduction. This would allow for the creation of a human being who is genetically identical to another.
Reproductive cloning
Broader use of the term “cloning.” Does not create a new genetically identical individual. Research includes therapy for human mitochondria disease and others that could replace damaged or diseased tissues without the risk of rejecting another’s tissue. Could create new skin tissue for burn patients.
Therapeutic cloning

19. 9/20/2018
What is cloning?
Multiple copies of genes or gene fragments, repeating nucleotide sequences
Others
Single cell organisms, like bacteria and fungi. This includes fermentation processes for production of bread, beer, and wine.
Entire plant asexual replication
Natural cloning occurs in sexual reproduction, when the embryo splits in two to produce twins.

20. Drug Resistance Gene Transferred by Plasmid
Drug Resistant Gene
mRNA
Plasmid
Resistant Strain
Plasmid gets out and
into the host cell
Enzyme
Hydrolyzing
Antibiotics
New Resistance Strain
Non-resistant Strain
Juang RH (2004) BCbasics

21. Target Genes Carried by Plasmid
Restriction
Enzyme
Restriction
Enzyme
Chromosomal DNA
DNA Recombination
Target Gene
Recombination
Transformation
1 plasmid
1 cell
Host Cells
Recombinant
Plasmid
Transformation
Juang RH (2004) BCbasics

22. 1 X100 X1,000 =100,000 Amplification and Screening of Target Gene
Plating
1 cell line, 1 colony
Plasmid
Duplication
X100
Bacteria
Duplication
X1,000
Pick the colony
containing target gene
=100,000
Juang RH (2004) BCbasics

23. Design of the Insert
Once you have your restriction enzymes chosen, it is time to design the final complete gene
The multiple cloning site (or whatever plasmid you are cloning into) should already have the 5’ portion of the gene intact (i.e. RBS, spacer, Met)
Sequences must be in frame
NcoI
BtgI
51 CTTTAATAAG GAGATATACC ATGGGCAGCA GCCATCACCA TCATCACCAC
M G S S H H H H H H
SacI AscI SbfI SalI NotI
BamHI EcoRI EcoICRI BssHII PstI AccI HindIII
101AGCCAGGATC CGAATTCGAG CTCGGCGCGC CTGCAGGTCG ACAAGCTTGC
S Q D P N S S S A R L Q V D K L A

24. Design of the Insert Multiple cloning site
71 ATGGGCAGCAGCCATCACCATCATCACCAC
M G S S H H H H H H
SacI AscI SbfI SalI
BamHI EcoRI EcoICRI PstI AccI HindIII
101AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC
S Q D P N S S S A R L Q V D K L A
The gene we want:
ggctgcgacagggcgagcccgtactgcggttaa
G C D R A S P Y C G *
Be aware of the amber stop codon: TAG
BamHI PstI
AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC
S Q D P N S S S A R L Q V D K L A
G C D R A S P Y C G *
ggctgcgacagggcgagcccgtactgcggttaa
AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA

25. Design of the Insert Translate the whole gene
Always check and re-check your sequence!
ATGGGCAGCA GCCATCACCA TCATCACCAC
AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA
Translate the whole gene
atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc
M G S S H H H H H H S Q D P G C D R A S ccgtactgcggttaactgcaggtcgacaa
P Y C G - L Q V D
Everything looks good: in frame the whole way!

26. Design of the Insert The wrong way to do it:
AGCCAGGATCC ggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAAGCTT
The gene is just inserted after the restriction site, which is out of frame with the plasmid-encoded start-codon/His-tag
atgggcagcagccatcaccatcatcaccacagccaggatccggctgcgacagggcgagcc
M G S S H H H H H H S Q D P A A T G R A cgtactgcggttaactgcaggtcgacaagctt
R T A V N C R S T S
Frame shifted = garbage!
**Some plasmids, for whatever reason, have restriction sites out of frame with the translated gene**

27. Finishing Touches
Restriction enzymes need 5’ and 3’ base pairs to cut properly
NEB has a reference guide for specific enzymes (see link below)
A good rule of thumb is 6 base pairs after the recognition site
Inserting a GC “clamp” at the end and beginning of the sequence is also a good idea
atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc
M G S S H H H H H H S Q D P G C D R A S ccgtactgcggttaactgcaggtcgacaa
P Y C G - L Q V D
Final gene, polished and ready to go:
gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc
S Q D P G C D R A S P Y C G - L Q V D

28. Design of the Primers
Once the insert is designed correctly, the next step is designing primers to order from IDT, based on insert synthesis strategy
Three main strategies towards insert synthesis:
PCR amplification
Klenow extension of overlapping primers
Complimentary full-length primers +
Insert
Vector

29. PCR Amplification of Insert from an Existing Gene
The most common method of insert synthesis
Necessitates a pre-existing construct
Extra restriction sites and/or amino acid residues can be added on each side of the gene
Internal mutations are more difficult
Insert

30. PCR Synthesis of Insert
PCR amplification from overlapping primers
No pre-existing construct is needed
PCR products messy, possibly making subsequent rxns difficult
Good for inserts >150 bp
F1: 10x 5’ 3’
F2: 1x 5’ 3’ 3’ 5’
R1: 1x 3’ 5’
R2: 10x
Insert
Full-length insert should still be the major product

31. Klenow Extension of Overlapping Primers
Two primers that are complimentary in their 3’ region are designed (overlap  15bp)
Extended to full length by the Klenow fragment of DNA Polymerase I
Useful if insert is 50 to 150 bp 5’ 3’ 3’ 5’
Insert 5’ 3’
Klenow
Klenow fragment: retains 3’ to 5’ polymerase activity, but does not have exonuclease activity

32. Complimentary Full-Length Primers
The simplest approach
Order two primers that compliment each other
Mix the two primers, heat, and anneal slowly (to ensure proper base-pairing)
Feasible if the total insert size is < 60 bp 5’ 3’
Anneal
Insert 3’ 5’

33. Designing Primers to Order
Once the insert synthesis technique is decided, primer design is fairly straight-forward
Forward primers:
Assess necessary overlap and copy the sequence from your designed gene, along with extra 5’ sequence
Reverse primers:
First, design exactly as if it were a forward primer: Copy necessary overlap and extra 3’ sequence from your designed gene
Once all this is in place, use pDRAW32 sequence manipulator to calculate the reverse compliment
Order the pDRAW32 calculated sequence directly

34. Cloning Out an Existing Gene
In the example mentioned previously, we would normally use full length overlapping primers, but let’s look at the more common case of having a preexisting gene:
Preexisting gene:
Overlap
tgcggcccagccggccatgggctgcgacagggcgagcccgtactgcggtggaggcggtgctgcagcgc
A A Q P A M G C D R A S P Y C G G G G A A A +
Goal gene:
gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc
S Q D P G C D R A S P Y C G - L Q V D
Extra sequence from gene design
gccagccaggatccgggctgcgacagg
ccgtactgcggttaactgcaggtcgacgc
Forward Primer:
Design of Reverse Primer:

35. Ordering Primers Forward primer to order: Design of Reverse Primer:
gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc
S Q D P G C D R A S P Y C G - L Q V D
Forward primer to order:
gccagccaggatccgggctgcgacagg
Design of Reverse Primer:
ccgtactgcggttaactgcaggtcgacgc
&
Reverse primer to order:
GCGTCGACCTGCAGTTAACCGCAGTACGG
Now we can order the primers:

36. Purification Tags and Selection (Anti-biotic Resistance)
Anti-biotic resistance (working concentration)
Ampicillin (100g/mL)
Kanamycin (35g/mL)
Tetracycline HCl (10g/mL)
Chloramphenicol (170g/mL in ethanol)

37. Digestion of Insert and Vector
Digest with the same restriction endonucleases
Optional (recommended) step:
Treat the plasmid DNA with Antarctic phosphatase
Decreases the background by stopping self-ligation of singly cut plasmid and background re-ligation

38. Ligation of the Insert into the Vector+
Ligation covalently attaches the vector and the insert via a phosphodiester bond (5’phosphate and 3’ hydroxyl of the next base)

39. Transformation
The functional construct is now ready to be transformed into new E. coli and grown up
The new DNA isolated from the E. coli must then be sequenced to make sure that everything worked
Once the sequence is confirmed, we are ready to go!

40. pBluescript origin of replication A widely used plasmid cloning vector
ampicillin
resistance
gene
MCS
MCS, Multiple Cloning Site

41. select for transformants with antibiotic
electroporation = colonies/g DNA
heat-shock = colonies/g DNA)

42. Identifying Recombinants
based on interruption of a gene
eg., lacZ gene = b-galactosidase
intact b-galactosidase produces blue color in presence of X-gal
-complementation or blue-white screening

43. Blue white screening
Screening by insertional inactivation of the lacZ gene
Lac promoter
MCS (Multiple cloning sites）
Ampr
pUC18
(3 kb)
lacZ’
ori
The insertion of a DNA fragment interrupts the ORF of lacZ’ gene, resulting in non-functional gene product that can not digest its substrate x-gal.

44. Recreated vector: blue transformants
Recombinant plasmid containing inserted DNA: white transformants
Recreated vector (no insert)
Recombinant plasmid (contain insert)
back

45. Multiple cloning sites
Multiple restriction sites enable the convenient insertion of target DNA into a vector
Ampr
ori
pUC18
(3 kb)
MCS (Multiple cloning sites）
Lac promoter
lacZ’
…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA…
. T h rA s n S er S e r Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser…
EcoRI
SacI
KpnI
SmaI
XmaI
BamHI
XbaI
SalI
HincII
AccI
PstI
SphI
Lac Z

51. Recombinant DNA / Genetic Engineering
The advent of recombinant DNA technology (gene cloning or manipulation) has dramatically broaden the spectrum of microbial genetic manipulation.
Based on the use of restriction enzyme (endonucleases) and DNA ligases as a means to cut & paste fragments of DNA
Foreign DNA fragments can be introduced into a vector molecule (eg. plasmid or bacteriophage), enable replication of the DNA in bacteria cell.
Recombinant technology in one form or another is used in many areas of biological research today.
Ability to modify and clone genes – accelerated the rate of discovery and the development of bioindustries

52. Recombinant DNA Technology
Applications
to study basic cellular mechanisms (eg.cell signaling pathway)
production of recombinant vaccine for therapy (cancer, genetic disorders, immune disorders, embryonic stem cells)
Production of recombinant proteins of medical & commercial value (eg. antibodies, insulin, RE)
Generate genetically modified / transgenic plants (GMOs plant) or animals with enhanced commercial and health properties.
Cloning of plants and animals.

53. What is DNA cloning?
isolation and manipulation of fragments of an organism’s genome by replicating independently as part an autonomous vector in another host species.
DNA fragment in vector will form recombinant DNA.

54. Applications of DNA cloning
DNA sequencing - genome & protein database.
Isolation & analysis of gene promoters / control sequences
Investigate protein / enzyme / RNA function by large-scale production of normal & altered forms
Identification of mutations -eg. gene defects cause disease
Biotechnology – large-scale commercial production of proteins & other molecules of biological importance (eg. human insulin & growth hormone)
Engineering animals & plants, gene therapy
Engineering proteins – altering properties

55. Basic steps in gene cloning
DNA
Vector
isolation
restriction
ligation
insert
Recombinant DNA
Host cells
Transformation/amplification
Selection / identification of clones
Validation of clones –analyses
RE, Southern blot, PCR, DNA sequencing
Positive recombinant DNA

56. The cloning of DNA in a plasmid

57. Recombinant DNA techniques used for Insulin production in E.coli
Isolate or cut the insulin gene from human DNA.
Restriction enzymes used to cut vector & insert for cloning
Ligate / paste insulin gene (insert) into a vector using DNA ligase
Recombinant plasmid DNA containing the insulin gene is transformed into E. coli host cells
Host cells multiply and produce one or more copies of the recombinant DNA. The insulin gene is now cloned
E. coli colony carrying the recombinant insulin is identified.
Recombinant plasmid DNA is isolated & analyzed for DNA sequencing
The insulin gene can be subsequently subcloned into an expression vector - for production of insulin in E.coli.

58. SUBCLONING
Simplest cloning experiment which uses many of the basic techniques
Involve the transfer of a fragment of cloned DNA from one vector into another
Use to investigate a short region of a large cloned fragment or to transfer a cloned gene into an expression vector

59. Steps in Sub-cloning Isolation of recombinant plasmid DNA
Digestion into discreet fragments
with restriction enzymes
Separation of fragments on
Agarose gel electrophoresis
Purification of desired target fragment
Ligation of fragment into a
new plasmid vector
Transformation and selection for
positive recombinant plasmid DNA