1. Dr. Peter John M.Phil, PhD Assistant Professor Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)

2. Cell Based DNA Cloning

3. Study of DNA Fragment
To amplify the desired DNA fragment to generate homogenous DNA population
DNA Cloning: To study the structure & function of DNA
Molecular Hybridization: The fragment of interest is not amplified but is detected within a complex mixture of many sequences.
To determine its chromosomal location/expression in a particular cell

4. DNA Cloning Isolate DNA from organism (e.g., extract DNA)
Cut DNA with restriction enzymes to a desired size.
Splice (or ligate) each piece of DNA into a cloning vector to create a recombinant DNA molecule.
Cloning vector = artificial DNA molecule capable of replicating in a host organism (e.g., bacteria).
Transform recombinant DNA (cloning vector + DNA fragment) into a host (e.g., bacteria) that will replicate and make copies.
E. coli is the most common host.

5. Cut DNA with restriction enzymes
Restriction enzymes recognize specific bases pair sequences in DNA called restriction sites and cleave the DNA by hydrolyzing the phosphodiester bond.
Cut occurs between the 3’ carbon of the first nucleotide and the phosphate of the next nucleotide.
Restriction fragment ends have 5’ phosphates & 3’ hydroxyls.
restriction
enzyme

6. Restriction enzymes
Most restriction enzymes occur naturally in bacteria.
Protect bacteria against viruses (bacteriophages) by cutting up viral DNA.
Bacteria protect their DNA by modifying possible restriction sites (methylation).
More than 400 restriction enzymes have been isolated.
Many restriction sites are palindromes of 4-, 6-, or 8-base pairs.
Short restriction site sequences occur more frequently in the genome than longer restriction site sequences.

7. Restriction Ends
Blunt ends: In a blunt-ended molecule both strands terminate in a base pair.
Overhang & Sticky ends: An overhang is a stretch of unpaired nucleotides in the end of a DNA molecule.
Sticky ends are useful for DNA cloning because complementary sequences anneal and can be joined directly by DNA ligase without using ‘adapters’.

10. Cut and ligate 2 different DNAs with EcoRI ---> recombinant DNA

11. Cell Based DNA Cloning Cell based DNA cloning:
Attaching DNA fragment to particle seq which are capable of independent replication.
Then recombinant DNA are transferred into host cell where they can replicate
Cell free DNA cloning:
By Polymerase Chain Reaction (PCR)

13. Principles of Cell based DNA cloning
Construction of Recombinant DNA molecule
Transformation in to host cell
Selective propagation of cell clones
Isolation of Recombinant DNA clones

15. Replicon & Host Cell Replicon:
A DNA/RNA molecule or a region of DNA/RNA, that replicate from a single origin of replication
Vector: Replicon used for cloning
Host Cell: Human, mammalian but mostly bacterial & fungal cells

16. Extrachromosomal replicons
Plasmids: Small circular dsDNA molecules, contain very few genes.
Bacteriophages:
Viruses which infect bacterial cells. These are dsDNA viruses which can be linear/circular
Various modification are made in naturally occurring replicons to be used as vector molecules

17. Intera & Intermolecular Association
When the fragments have same type of overhang different types of associations can occur.
Intramolecular: B/W two vector molecules/cyclization
Intermolecular: B/W target DNA.

18. Intera & Intermolecular Association
How to avoid Intera & intermolecular association
Cutting the vector with two different restriction endonuclease
Vector dephosphorylation: by alkaline phosphatase 5’ end phosphate can be removed, which avoid recircularization

20. DNA Cloning in bacterial cells
Transformation:  Genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane
Transduction: Genetic alterations resulting from introduction of DNA by viruses
Transfection: The process of deliberately introducing naked or purified nucleic acids by non-viral methods into eukaryotic cells

22. Plasmid Vectors Modifications
Insertion of multiple cloning site polylinker
Insertion of an antibiotic resistant gene
Insertion of selection system for screening of recombinants

23. Screening transformed cells
Antibiotic resistant gene: Host cell is chosen which is sensitive to certain particular antibiotic, but vector molecule contain a gene so, transformed cell can survive
β-galactoside gene complementation: The host cell & vector contain fragments of β-galactoside gene (transformed cell convert x-gal into blue color)

24. Recombinant Screening
Selectable marker is ampR. Ampicillin in growth medium prevents growth of all other E. coli that do not contain plasmid.
Cluster of several different restriction sites called a polylinker occurs in the lacZ (-galactosidase) gene.
Cloned DNA disrupts reading frame and -galactosidase production.
Add X-gal to medium; turns blue in presence of -galactosidase.
Plaque growth: blue = no inserted DNA and white = inserted DNA.
Some % of digested vectors will reanneal with no insert. Remove 5’ phosphates with alkaline phosphatase to prevent recircularization (this also eliminates some blue plaques).

26. DNA libraries
DNA library is a collection of cloned DNA fragments.  There are two types of DNA library
The genomic library: contains DNA fragments representing the entire genome of an organism
The cDNA library: contains only complementary DNA molecules synthesized from mRNA molecules in a cell. 

27. Genomic DNA library

28. cDNA library
Extract total RNA from a specific tissue/development stage
Convert RNA into cDNA
Ligate to vector

29. cDNA library

30. Restriction Mapping for DNA study
Restriction mapping of DNA involves cutting the DNA with one or more of a series of different restriction nucleases and separating the resulting fragments according to the size by gel electrophoresis

32. Thanks