1. Chapt. 4 Molecular Cloning Methods
Learning outcomes:
Explain major tools of recombinant DNA technology – date from 1970s
Expand understanding of new tools
Impt. Figs: 1, 2, 3, 4, 5, 10,
11, 12, 15*, 16, 17, 18, 20,
23, 24, 25, 26; Table 1
Review Q: 2, 4*, 6, 7, 8, 9*,
10, 11, 13*, 14*; AQ 1

2. 4.1 Gene Cloning in Bacteria
Link prokaryotic or eukaryotic genes to small bacterial DNAs
(plasmids or phage)
Insert recombinant molecules into bacterial hosts to replicate
Can produce large quantities of these genes (or proteins) in pure form
Fig. 4

3. Type II Restriction Endonucleases
Discovered late ‘60s
Named for organism
Recognize specific DNA sequence, cut ONLY at that sequence
Recognize 4-bp to 8-bp sequences
Frequency of cuts less when recognition sequence is longer: (1/4)n
(size of piece larger ~4n)

4. Restriction Endonucleases, DNA Ligase
Many make staggered cuts in DNA strands
Single-stranded overhangs, sticky ends, can base-pair together (5’-PO4, 3’-OH ends)
Makes joining 2 different DNA molecules easier
DNA ligase needs 5-PO4, 3’-OH ends + ATP
Recognition sequence
usually symmetrical
palindromes
EcoRI: 5’-GAATTC-3’
3’-CTTAAG-5’

5. Restriction-Modification System
Enzymes cut foreign DNA, protect cells
What prevents enzymes from cutting up host DNA?
paired methylases recognize, methylate, same site
Form restriction-modification system, R-M system
Methylation protects DNA; after replication, parental strand is partially methylated
Fig. 1

6. Early Gene Cloning – recombinant DNA
Boyer & Cohen used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of host chromosome
Each plasmid had site for EcoRI
Linear DNA with same sticky ends
Sticky ends base pair
Some ends re-close
Others join 2 pieces
DNA ligase joins 2 pieces with covalent bonds
Transform E. coli; select drugr
Fig. 2

7. Vectors
Vectors function as DNA carriers, permit replication of recombinant DNAs
Typically 1 vector, 1 piece of foreign DNA
Need vector for replication
Foreign DNA has no origin of replication,
(site where DNA replication begins)
Prokaryotic vectors:
Plasmids; Phages
Selectable marker,
Restriction sites for cloning
Origin of replication
pBR322

8. pBR322 Cloning Clone foreign DNA into PstI site of pBR322
Cut vector to generate sticky ends with PstI
Cut foreign DNA with PstI
Combine vector and foreign DNA: DNA ligase seals
Transform E. coli
(CaCl2, electroporate)
Select Tetr
Fig. 4

9. Screening Transformants pBR322
Transformation yields bacteria :
Religated plasmid
Religated insert
Recombinants
Identify transformants, recombinants with antibiotic resistance
In tetracycline, only cells with plasmid grow, not just foreign DNA pieces
Test copies of original colonies with ampicillin (kills cells with plasmid including foreign DNA)
Identify desired recombinants
Fig. 4

10. Screen With Replica Plating
Transfer copies of clones from original tetracycline plate to plate with ampicillin
Transfer with sterile velvet
Desired recombinant colonies do NOT grow on ampicillin plate
Pick clone from original plate
Fig. 5

11. pUC plasmid has b-galactosidase fragment
Ampicillin resistance gene
MCS in lacZ’ (N-terminal a-peptide of b-galactosidase)
Foreign DNA in MCS disrupts ability to make b-gal a-peptide:
On X-gal media: clones producing functional b-gal enzyme (from a and host b peptide) produce blue colonies; clones with insert are white.
Fig. 6
Fig. 6

12. Summary Basic Plasmid Cloning Vectors
pBR322 and the pUC plasmids
pBR322 has
2 antibiotic resistance genes
Many unique restriction sites for inserting foreign DNA
Most of these sites interrupt antibiotic resistance, making screening straightforward
pUC has
Ampicillin resistance gene
MCS that interrupts the b-galactosidase gene a-peptide
MCS also facilitates directional cloning, using pieces cut with 2 different restriction enzymes

13. Phages As Vectors
Bacteriophages: natural vectors transduce bacterial DNA from one cell to another
Phage vectors infect cells much more
efficiently than plasmids transform cells
Clones are not colonies of cells, but rather plaques,
clearing of bacterial lawn due to
phage infect, kill bacteria in area
lambda

14. Lambda (l) Phage Vectors
Replacement vectors:
Removed middle region (needed only for lysogeny)
Retained genes for phage replication
Replace removed phage genes with foreign DNA
Phage vectors hold larger amounts of foreign DNA
Accept up to 20-kb of DNA
Traditional plasmid vectors usually less
Lambda phage vectors require minimum size DNA (12 kb) insert to package into phage particle

15. Cloning Using a Lambda Phage Vector
Join DNA in vitro
Add packaging
proteins from cell
extract
Infect cells
Fig. 8

16. Genomic Libraries
Large number of clones: all genes from species’ genome
Restriction fragments can be packaged into phage, about 16 – 20 kb per fragment (often partial digests)
Need thousands of clones for library:
ex. Yeast genome 107 bp/ 104 bp/clone -> ____ clones
ex. Human genome 3 x 109 bp /104 bp/clone -> ___
Once library is established, search for gene of interest (BL 415 experiment with plasmid library)

17. Plaque Hybridization
Search genomic library with probe to identify clone containing desired gene
Ideal probe – labeled nucleic acid with sequence matching the gene of interest
Fig. 9

18. Cosmids Cosmids designed to clone large DNA fragments
Behave as plasmid and phage
Contain
cos sites, cohesive ends of l phage DNA, allow DNA to be packaged into l phage head
Plasmid origin of replication permits replication as a plasmid in bacteria
Most l genome removed so hold large inserts (40-50 kb)
So little phage DNA, can’t replicate like phage; infectious, carry recombinant DNA into bacterial cell, then plasmid

19. M13 Phage Vectors Long, thin, filamentous phage M13
Normal size 6.4 kb (10 genes)
Modified vector contains:
Gene fragment with b-galactosidase
Multiple cloning site like pUC plasmid
Advantages
Can hold large DNA; length of phage increases
Phage genome is single-stranded DNA
Fragments cloned (into ds intracellular form) are recovered in single-stranded form
Phage does not kill cells; extrudes particles
Useful for sequencing DNA, mutagenesis

20. M13 Cloning permits recovery of Single-stranded DNA
After infecting E. coli, single-stranded phage DNA was converted to double-stranded replicative form (RF, plasmid-like)
Purify replicative form to clone foreign DNA into MCS; ligate vitro
Transform recombinant DNA into host cells, results in plasmid-like RF molecule in cells
Phage particles, contain one single-stranded DNA (+ strand), secreted from transformed cells, can be collected from media
Fig. 10

21. Phagemids are versatile Vectors: pBluescript (pBSK)
Fig. 11
Aspects of both phages and plasmids:
Ampr for selection of clones
MCS inserted into lacZ’ gene permit blue/white screening
Origin of replication of single-stranded phage f1 permits recovery of single-stranded recombinant DNA (like M13)
MCS has phage RNA polymerase promoters, 1 on each side of MCS; permits specific RNA transcripts in vitro

22. Eukaryotic Vectors, High Capacity vectors
Plasmids and Viruses
clone genes in eukaryotic cells
Plasmid shuttle vectors (origins of replication for both bacterial and eukaryotic host cell; ex. Yeast YEP24
Selectable markers in both hosts; MCS sites
Yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) clone huge pieces
Vectors based on Ti plasmid for plant cells

23. Identifying specific clone with specific probe
Probes can identify a desired clone from among thousands of irrelevant ones:
Two types widely used:
Recognize gene or mRNA with Oligonucleotides: Short RNA or DNA sequences that– hybridize
Can use homologous gene from another organism
If amino acid sequence is known, deduce possible nucleotide sequences to code for these amino acids
Recognize specific protein product with Antibodies

24. cDNA Cloning from Eukaryotes into Bacteria
cDNA: complementary DNA or copy DNA
made from mRNA
A cDNA library: set of clones representing many mRNAs from cell type or tissue or time
Library can contain thousands of different clones
* Need cDNA clones because genomic clones have introns, aren’t expressed in bacteria

25. Making cDNA Library from eukaryotic mRNA
Reverse Transcriptase (RT)
mRNA -> DNA
Primer
oligo(dT) to polyA at 3’ end
RNAse H degrades RNA
DNA polymerase Pol I finishes
Terminal transferase to add C tails and G tails for cloning
Fig. 12

26. Vector choice for cDNA cloning
Purpose and method to detect positive clones
If cloning gene and small fragment, plasmid or phagemid like pUC or pBS used, with colony hybridization and labeled DNA probe
If large fragments, use cosmids, or l phage or YAC, BAC
For expression of gene product, use cDNA of eukaryote cloned in pUC, phagemids of lambda
under control of lac promoter for transcription and translation of gene; use hybridization, antibody screening (more later)

27. Summary clone cDNA into Bacteria
Make cDNA library with synthesis of cDNAs one strand at a time
Use mRNAs from a cell as templates for 1st strands, then 1st strand as template for 2nd
Reverse transcriptase generates 1st strand
DNA polymerase I generates 2nd strand
Give cDNAs oligonucleotide tails that base-pair with complementary tails on a cloning vector
Use recombinant DNAs to transform bacteria
Detect clones with:
Colony hybridization using labeled probes
Antibodies if gene product is translated

28. Polymerase chain reaction (PCR): amplifies DNA exponentially; yields DNA fragments for cloning, forensics, diagnostics
Fig. 15

29. Standard PCR Invented by Kary Mullis in 1980s
DNA polymerase copies selected region of DNA
Short DNA primers hybridize to DNA on either side of piece of interest – initiate DNA synthesis through that area, X
Selected region DNA doubles in amount each cycle
Special heat-stable polymerases work after high temperatures that separate (denature) strands simplifies process – ex. Taq polymerase
Real-time PCR uses fluorescent dyes to quantify amplification, reflect amount of mRNA or DNA in cells
(Fig. 17)

30. Reverse transcriptase PCR (RT-PCR) to clone specific cDNA
Start with mRNA, not dsDNA
Convert mRNA to DNA with primer specific to gene
Use forward primer to convert ssDNA to dsDNA
Then standard PCR
With primer choice, restriction enzyme sites added to cDNA
2 sticky ends permit directional cloning – good for protein expression
Fig. 16

31. 4.3 Expressing Cloned Genes
Cloned gene permits production of large quantities of particular DNA sequence
Expression vectors yield protein products
Strong promoter, ribosome binding site near ATG codon
Make as pure protein, or as fusion to bacterial peptide
Regulated expression:
Inducible expression vectors keep cloned gene repressed until time to express:
Large quantities of eukaryotic protein can be toxic

32. Fusion proteins: Oligo-histidine (His-tagged) Expression Vector
Sequence upstream of MCS encodes 6 His which get added to protein:
Oligo-histidine high affinity for divalent metal ions (Ni2+)
Purify protein by nickel affinity chromatography
His tag removed using enzyme enterokinase if needed
Fig. 20

33. Fusion Proteins with lacZ’
Cloning vectors (pUC and pBS) are expression vectors using lac promoter with lacZ’ a-peptide
If inserted DNA in same reading frame as interrupted gene, fusion protein results
Partial b-galactosidase at N- end
Inserted protein at C-end
Fusions often more stable
Fusions also assist detection, purification
Fig. 18

34. Regulation: lac Promoter/ operator
lac promoter inducible with lactose or synthetic IPTG; binds repressor and removes it from O
Transcription stays off until induced

35. Fusion Proteins also in lgt11 vector
Phage contains lac control region and lacZ’ gene (a-peptide)
Products of gene correctly inserted are fusion proteins with b-galactosidase peptide
Use blue-white screening on host with b-peptide
Fusion reduces degradation of foreign protein
Fig. 21

36. Antibody screening with lgt11 (or plasmids)
Lambda phages with cDNA inserts on E. coli
Proteins released are blotted onto membrane
Probe with specific 1o antibody to protein
Antibody bound to protein from plaque is detected with labeled 2o antibody
Fig. 22

37. Summary – Expression in Bacteria
Expression vectors can produce fusion proteins
One part from coding sequences in vector
Other part from sequences in cloned gene
Many fusion proteins can be simply purified by affinity chromatography (His-tag; Flag-tag; TAP-tag)
Fusion proteins can be detected with antibodies after blotting of colonies or plaques:

38. Bacterial Expression System Shortcomings
Bacteria recognize proteins as foreign and destroy
Posttranslational modifications (glycosylation, phosphorylation) are not done correctly
Bacterial cytoplasm not permit correct protein folding
High levels of cloned eukaryotic proteins can be expressed in useless, insoluble (inclusion) form

39. Use Eukaryotic Expression Systems
Initial cloning in E. coli using shuttle vector, that replicates in both bacterial and eukaryotic cells
Ex. Yeast Saccharomyces cerevisiae well-suited :
Rapid growth, ease of culture
Multicopy plasmids (from natural 2 m plasmids)
Eukaryote, so more appropriate posttranslational modifications to expressed proteins
Can secrete protein for easy purification
Ex. Plasmid YEP24 shown earlier

40. Ex. Baculovirus expression vectors; transform (transfect) insect cells in culture
Large circular DNA genome;
Major viral structural protein made in huge amounts
Strong promoter for polyhedrin protein
Produce lots of recombinant protein per liter of medium
Non-recombinant viral DNA do not replicate because lack essential gene
Fig. 23

41. Methods for Transfection of animal cells with vectors for clones or protein production
[Transformation for animal cells used for cancer cells]
Calcium phosphate
Mix cells with DNA in phosphate buffer
Solution of calcium salt added to form precipitate
Cells take up calcium phosphate crystals, includes DNA
Electroporation
Electric shock for DNA to enter cells
Liposomes
DNA mixed with lipid to form liposomes, small vesicles
Liposomes fuse with cell membrane, carry DNA inside

42. Foreign genes can be cloned and expressed in eukaryotic cells
Summary
Foreign genes can be cloned and expressed in eukaryotic cells
Eukaryotic systems have some advantages:
Proteins tend to fold properly, are soluble rather than aggregated into insoluble inclusion bodies
Posttranslational modifications (phosphorylation, glycosylation) made in eukaryotic manner
Can use transient clones for tests gene function
Select stable clones expressing transgene (as for construction transgenic organisms)

43. Review problems
2. Why does one need to attach DNAs to vectors to clone them?
Diagram the process of cloning a DNA fragment into BamHI and PstI sites of vector pUC18. How would you screen for clones that contain an insert?
Explain the blue/white lacZ’ detection system
7. You want to make a genomic library with DNA fragments averaging about 45 kb in length. Which vector discussed in this chapter would be most appropriate? Why?

44. Review problems 11. Diagram a method for creating a cDNA library.
Outline the polymerase chain reaction (PCR) method for amplifying a given stretch of DNA.
What is the difference between reverse transcriptase PCR (RT-PCR) and standard PCR? For what purpose would you use RT-PCR?
For what purpose might you want cDNA vs genomic library?

45. Review problems
AQ 1. Given an amino acid sequence of part of a protein from a hypothetical gene you want to clone:
Pro-arg-tyr-met-cys-trp-ile-leu-met-ser
Look at the code chart
What sequence of 5 aa would give a 14-mer probe with the least degeneracy for probing a library to find gene of interest?
How many different 14-mers would you need to make to be sure probe matches sequence in cloned gene perfectly?
lf you started your probe one aa to the left of the one you chose in (a), how many different 14-mers would you have to make?