1. Molecular Cloning

2. Definitions Cloning : Sub-cloning: Recombinant Plasmid :
Obtaining a piece of DNA from its original source (Genome) and introducing it in a DNA vector
Sub-cloning:
Transfer of a cloned DNA insert or a part thereof from one vector to another vector
Recombinant Plasmid :
Vector into which foreign DNA was introduced
Recombinant organism
Organism with a recombinant vector

3. Why clone?
Separate, identify, manipulate or express a specific DNA fragment 3

4. Step 1- Separate Two approaches: Fragment/digest genomic DNA
Generates a vast number of fragments
May be difficult to find fragment of interest
PCR Amplification
Much less fragments
Much easier to find sequence of interest

5. DNA Replication & Amplification
The Polymerase Chain Reaction

6. Polymerases Primer -OH 3’OH end Polymerase 5’…GTACT
3’…CATGAATGCTGCATTTGCGGGCATTACTC…5’
TACGACGTAAACGCCCGTAATGAG
DNA or RNA
Template

7. The Polymerase Chain Reaction-PCR
Repetitive replication of a given region of DNA
Allows the exponential amplification of a given region of DNA
Increases the relative representation of the region of interest
Allows the isolation of a given region of DNA 7

8. PCR-1st Cycle <3’GGAACGGTACCGT5’
Denaturation (95oC)
Annealing of primers (Tm)
<3’GGAACGGTACCGT5’
5’CATACCGTGGGGTGCA………..ACGCGTTGCGATGGCA3’
3’GTATGGCACCCCACGA………..TGCGCAACGCTACCGT5’
5’CCGTGGGGT3’>

9. 5’CATACCGTGGGGTGCA………..ACGCGTTGCGATGGCA3’
Extension (72oC)
<3’GGAACGGTACCGT5’
5’CATACCGTGGGGTGCA………..ACGCGTTGCGATGGCA3’
3’GTATGGCACCCCACGA………..TGCGCAACGCTACCGT5’ 3’ 5’  
5’CCGTGGGGT3’> 9

10. PCR-2nd Cycle 3’ 5’ ---------------------------------- 5’ 3’ 3’ 5’
Denaturation  
Annealing
/Extension

11. PCR-Subsequent Cycles
Only this template is amplified exponentially: 2n times
32 times total

12. Review of PCR Cycles PCR Primers:
Short single stranded nucleotide sequences complementary to the targets
15-30 nucleotides
Used in excess as compared to target to favor primer annealing rather than template self annealing

13. Review of PCR Cycles Annealing: Extension:
Temperature at which primers anneal to complementary target sequences
Must be below primer Tm
Must be a temperature that allows both primers to anneal
Usually between 55-75oC
Extension:
Carried out at temperature optimum for DNA polymerase
Usually 72-75oC for Taq polymerase

14. Primers Characteristics:
Short oligonucleotides complementary to sequences that flank the region of interest
Establish the point of initiation of replication
Establish the point of termination of replication 14

15. Primer Design 5’ complementarity 3’…………….ATGGGTATTGGCC…………………..-5’
Template
CCATAACCGG-OH3’
5’CGA
3’ complementarity
3’…………….ATGGGTATTGGCC…………………..-5’
Template
TACCCATAACC
TA-OH3’ 15

16. Primer Design 5’ 3’ 3’ 5’ 3’ 3’ Region of interest 3’
Correct orientation
Wrong orientation 16

17. Problem 1- AAAAAA 2- TTTTTT 3- GGGGGG 4- CCCCCC
5’-AAAAAAAAAAAA
GGGGGGGGGGGGG-3’
You wish to amplify the sequence represented by the box. Which primer pair represent the correct orientation to accomplish this?
1- AAAAAA
2- TTTTTT
3- GGGGGG
4- CCCCCC 17

18. Utility of PCR
Amplification and isolation of a given region by changing its relative representation
Between 100bp and 10Kpb
Screening to determine the presence of a sequence of interest
Presence or absence of an amplification product
Site directed mutagenesis
Used to add or remove nucleotides from the original template 18

19. Cloning Generate compatible ends DNA ligation Restriction Enzyme
Appropriate vector
Fragment of interest
DNA Recombination
Intermolecular ligation
Recombinant
Intramolecular ligation
Non-Recombinant
Transformation
Host Cells
Recombinant cell
Non-Recombinant cell
1 plasmid/1 cell

20. Amplification of Recombinant Plasmids
Duplication
1 colony= 1 clone with 1 plasmid + 1 insert
Bacterial growth

21. Screening and Identification of Recombinant Plasmid clones
Blue-white screening
Restriction mapping
Hybridization
PCR

22. Screening for Recombinants
Alpha complementation
“Blue-white" screening
β-galactosidase (LacZ)
α-peptide (aa 1-92) - Inactive
ω-peptide (remainder of LacZ) –Inactive
α + ω peptides  active β-galactosidase

23. Alpha complementation
Used in many vectors
LacZ promoter
MCS - restriction sites for cloning
5' end of lacZ gene - LacZ α-peptide
Distinguish E. coli with:
Empty vector
Vector + insert DNA

24. Blue – White Screening E.coli → LacZ ω pUC19 → LacZ α
Medium with X-gal
E.coli - LacZ ω + plasmid with insert
NO LacZ α → white colonies
E.coli - LacZ ω + plasmid without insert
LacZ α → blue colonies