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
Yield: N=No(2n)
N: Final number or mass
No: Initial number or mass
32 times total

12. Problem
You wish to amplify a 1kb sequence from 10ng of a 10 kb single stranded template with primers spanning positions and How many cycles are required to obtain 1µg of the desired product?

13. Solution Determine initial mass of desired product
10kb = 10ng therefore 1kb = 1ng or 10-3µg (Single stranded)
Double stranded would be 2 X 10-3 µg
Determine number of cycles starting from initial mass of desired product
1µg = 2 X 10-3µg(2n); solve for n
Approx. 9
Determine number of cycles to obtain double stranded desired product
3 cycles
Total: 9+3 = 12

14. 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

15. 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

16. 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 16

17. Primer Design 5’ complementarity 3’…………….ATGGGTATTGGCC…………………..-5’
Template
CCATAACCGG-OH3’
5’CGA
3’ complementarity
3’…………….ATGGGTATTGGCC…………………..-5’
Template
TACCCATAACC
TA-OH3’ 17

18. Primer Design 5’ 3’ 3’ 5’ 3’ 3’ Region of interest 3’
Correct orientation
Wrong orientation 18

19. Primers 2 primers are required for exponential amplification
Forward primer
Primer whose sequence is the same as that of the template indicated
Reverse primer
Primer whose sequence is complementary to that of the template indicated
Example 3’-GCGGTT••••••••••ATCGTTA-5’
Forward primer: ATTGCTA
Reverse primer: CGCCAA

20. 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 20

21. 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 21

22. 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

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

24. Screening and Identification of Recombinant Plasmid clones
Restriction mapping
PCR