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

Cell Based DNA Cloning

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

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.

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

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.

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’.

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

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)

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

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

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

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.

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

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

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

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)

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).

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. 

Genomic DNA library

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

cDNA library

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

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