Molecular Biology Techniques: Cloning and DNA

Molecular Biology Techniques

• Uses the manipulation of enzymes to investigate DNA.
• Applied to genes to DNA.

Genes to DNA

• DNA is the genetic material.
• Isolation and manipulation of DNA allows:
  • Isolate and modify genes to understand their function.
  • Shift from mutant phenotype to gene sequence.
  • Manufacture proteins like insulin and factor VIII.
  • Generate vaccines.
  • Create transgenic plants or organisms.
  • Diagnose genetic diseases.
  • Develop gene therapy.

Recombinant DNA Technology

• Allows for the isolation and manipulation of DNA, also known as gene cloning.
• Methods isolate sections of DNA and then create multiple copies of these sections.
• Gene cloning refers to the many copies or clones made of unique pieces of DNA.

Molecular Cloning

• After reducing a genome to smaller pieces of DNA, these pieces must be reproduced in large amounts for further study.
• Isolated DNA fragments such as the coding sequence of a gene, are inserted into a cloning vector, like a plasmid, to create a recombinant DNA molecule.
• A cloning vector is a DNA molecule that, once introduced into a host organism, can self-replicate.
• The recombinant DNA molecule is introduced into a biological system that replicates the DNA to make many copies.
• Identical copies resulting from this process are called DNA or molecular clones.

Restriction Enzymes

• Function as molecular scissors to cut DNA.
• Recognize specific nucleotide sequences in the DNA and cut both strands of the sugar-phosphate backbone.
• First identified in bacteria, they protect against viral infection by cutting, or restricting, viral DNA.
• Different restriction enzymes recognize different nucleotide sequences, where the sequence forms a palindrome, reading the same on both strands in the 5'-3' direction.
• Restriction enzymes cut with a characteristic cleavage to leave cohesive, or sticky, or blunt ends.
• Restriction enzymes cut genomes to produce many DNA fragments.
• EcoRI cuts the human genome into roughly 730,000 pieces.
• Restriction site occurrence depends on the percentage of GC or AT content in the genome.
• DNA sequences are not random in the genome, so restriction sites can be closer or further apart in different regions.
• The number and size of fragments depends on the restriction enzyme, genome size, and nucleotide abundance.
• Four cutters cut more frequently than six cutters.

Genomic Digest

• Cuts DNA into different fragments.
• Drosophila genomic DNA that is digested with EcoRI, lanes 1-10, can be separated on an agarose gel.
• It appears as a smear, as EcoRI cuts DNA into fragments with sizes varying from tens to thousands of base pairs in length.
• Each fragment is known as a restriction fragment.

Restriction Enzyme Mapping

• Restriction maps are made by cutting DNA with individual restriction enzymes.
• Agarose gel electrophoresis separates digested DNA samples, allowing separation of DNA fragments by size.
• The size of the cut DNA sequence can be identified by comparing how far they move in the gel to DNA fragments of known sizes.
• Comparison shows recognition sites relative to one another and how often the enzymes cut the DNA.
• Different restriction enzymes are used in pairs to identify locations.

Restriction Enzyme Map

• Required for identifying locations of genes aiding in the study and following gene manipulation.
• Necessary to generate restriction enzyme maps' of plasmids and the genome.
• Enzyme A, cuts three times resulting in there being three pieces.

Recombinant DNA

• Large amounts of individual DNA fragments are needed to allow for further study or manipulation.
• Bacteria or yeast are used to replicate and amplify individual DNA fragments.
• A mechanism is needed to introduce DNA into the host organism.
• Plasmids or circular DNA molecules work as cloning vectors to carry DNA fragments into the host, meaning bacteria.

Plasmids

• Contain a replication origin that allows replication independently of the bacterial chromosome.
• High copy numbers of plasmids, around 100-200, can be maintained in each bacterium.
• Contain antibiotic or AB resistance genes, allowing the selective growth of bacteria with plasmids.
• DNA cloning plasmids also include a Multiple Cloning Sequence or MCS.
• There are different types - cloning plasmids for cloning genes and expression plasmids adapted to allow gene expression.

Cloning DNA Molecules

• A DNA molecule can be cloned by inserting it into a plasmid cloning vector.
• Cut the plasmid with a restriction enzyme and cut the DNA fragment, which have the same overhang sequences, and then pair it using enzyme ligase.

Creating Recombinant DNA Molecules

• DNA cut by the same restriction enzyme will have the same cohesive ends.
• These cohesive ends have complementary base pairs.
• Complementary base pairs will anneal to one another, happening due to base pairs forming hydrogen bonds A-T, C-G.
• The free 3' OH and 5' PO4 groups are covalently joined by DNA ligase.

Cloning DNA

• To clone a DNA fragment, the fragment and cloning vector, like the plasmid, are cut with the same restriction enzyme.
• Both the vector and fragment have complementary cohesive ends.
• Cut DNA fragments and the cut vector are mixed together.
• DNA fragments will anneal to cohesive ends in the vector.
• DNA ligase will ligate the DNA fragment and cloning vector together to form a recombinant DNA molecule.

Cloned DNA Amplification

• Happens in E. coli, individual recombinant plasmids can be taken up by the bacteria.
• Using the origin of replication or ori, 100-200 copies of the recombinant plasmid are generated in each bacterium.
• As bacteria divide, each one harbors 100-200 copies of the recombinant plasmid.
• Plasmids used for DNA cloning have individual restriction sites clustered in a multiple cloning site or MCS, where DNA inserts are cloned.

Identifying Recombinant Plasmids

• In many cloning plasmid vectors, the MCS is placed within the lacZ gene.
• lacZ encodes B-galactosidase, which acts on X-gal to produce blue bacteria.
• The lacZ gene is disrupted when an insert is cloned.
• Recombinant plasmids that contain an insert no longer make B-galactosidase, so they cannot cleave X-gal and generate white colonies.

Uses of Cloned DNA I

• Mapping and sequencing of genes within the genome.
• Clone and piece together genomic DNA.

Genomic DNA Library

• Often done by creation
• Used to identify changes in the genome with specific phenotypes or diseases.
• Characterize how the genome is organized and the location of repetitive DNA.
• Genetically engineer organisms by transferring genes between organisms.
• Produce large amounts of proteins from isolated genes for antigens in vaccines or therapeutics.

Cloning Eukaryotic Genes

• To express a eukaryotic protein for study or use in therapies.
• Genomic DNA cannot be used for this purpose because it contains introns.
• The eukaryotic genome contains many genes, 20,000 to 50,000, and most contain introns.
• Introns can make eukaryote genes range from 10 to 100kb.
• Isolating mRNA, where introns are removed, is required to convert this back to DNA to create CDNA.

Eukaryotic Gene Expression

• Different tissues or cells express different genes.
• Heart cells express genes for heart function, and kidney cells express genes for kidney function only.
• Harvesting mRNA from different cells allows discovery of which genes are expressed by which cells.

CDNA

• Required to express eukaryotic proteins or identify genes that are expressed in a specific cell type, and requires making DNA copies of the transcribed mRNA.
• mRNA is produced when genes are transcribed.
• mRNA does not contain introns.
• Can't clone mRNA because it is only DNA.
• Complementary DNA or cDNA, involves a DNA copy of mRNA produced using reverse transcriptase.
• cDNA libraries contain complementary DNA copies of the mRNAs in a cell population representing the genes expressed in the population where the mRNA is harvested.
• cDNAs are used in eukaryotic expression vectors as introns are removed.

cDNA Libraries

• Each mRNA from a sample is cloned as a cDNA in a plasmid.
• A cDNA library consists of a large collection of plasmids which each contain a single cDNA.
• Genes that are highly expressed in the cells from which the mRNA was isolated will be more present in the library than genes that are expressed at low levels.
• Genes not expressed in the tissue will not be present in the library.
• Regulatory sequences and introns are not present in cloned cDNA libraries.

Expression Plasmids

• Used to allow gene expression.
• Expression plasmids are plasmid cloning vectors designed to allow expression of cloned genes in bacteria to produce large amounts of encoded proteins, like insulin, in a host cell.
• Expression plasmids need key bacterial sequences to directly transcribe and translate the cloned DNA.
• Eukaryotic expression plasmids are plasmid cloning vectors needed to express cloned genes in eukaryotic tissue culture cells.
• Eukaryotic expression plasmids contain key eukaryotic regulatory sequences that directly transcribe and translate the cloned DNA.

Cloning Vector Vs Expression Vector

• Cloning vectors are small pieces of DNA that can be maintained in bacteria.
  • They introduce cloned DNA into bacteria to make many copies of the insert DNA.
  • They can be plasmids, cosmids, phages, or BACs.
  • They include a replication origin, unique restriction sites, and an antibiotic resistance gene.
• Expression plasmids introduce cloned DNA into the bacteria, allowing expression of the cloned gene.
  • Used to obtain the gene product of the cloned DNA, which might be a protein or RNA.
  • Can only be plasmids.
  • Include all features of a cloning plasmid, in addition to to regulatory elements such as promoters, transcription initiators, and translation initiation sites.

DNA Cloning

• Can allow the production of therapeutics, such as Human Insulin Production.

Nucleic Acid Hybridization

• A method to identify nucleic acid fragments or clones that contain specific sequences.
• Relies on the complementarity of nucleic acid strands.
• Complementary strands of nucleic acid anneal to one another.
• Can be used to identify DNA or RNA fragments containing specific sequences.

Nucleic Acid Hybridization Principle

• The individual strands of DNA molecules can be separated by heating or in alkali conditions.
• Cooling or neutralizing complementary strands will anneal to one another.
• Complementary strands hybridize to one another.

Nucleic Acid Hybridization

• Single strands of DNA hybridize to complementary RNA molecules.
• ssDNA can be used to identify complementary fragments of RNA or DNA.

Nucleic Acid Hybridization Technique

• Can be used to identify DNA or RNA that matches a specific sequence.
• ssDNA can be used as a probe to identify specific DNA fragments or clones in a collection that have a sequence complementary to the probe DNA.

DNA Probe Preparation

• DNA must be labeled before it can be used as a probe.
• Allows for after-the-fact detection of the DNA probe.
• Labeled using DNA polymerase, which incorporates labeled dNTPs.
• dNTPs can be labeled using radioactive 32P or by attaching a fluorescent molecule or attaching digoxygenin (steroid molecule).

Identifying DNA Fragments

• Nucleic acid hybridization identifies DNA fragments in the genome containing genes.
• A technique known as Southern hybridization, or Southern blot, named after Ed Southern who invented the method.
• Allows identification of which DNA fragment contains a specific gene or sequence after genomic DNA is digested with a specific restriction enzyme.

Southern Blot

• DNA fragments are separated by gel electrophoresis then denatured by soaking the gel in alkali.
• The DNA fragments are transferred to a DNA-binding membrane made of nitrocellulose or nylon.
• A single-stranded labeled DNA probe, made from a specific sequence of interest, is placed in a hybridization solution, together with the membrane bound with DNA fragments.
• The labeled probe DNA hybridizes to the matching complementary DNA fragment on the DNA membrane.
• The position of the complementary DNA fragment is identified as it is labeled by the probe.

Genomic Southern Blot

• Allows identification of genomic DNA fragments generated by restriction enzymes, called restriction fragments, containing specific DNA sequences.
• The restriction fragment is cloned into a plasmid for analysis and manipulation.

DNA Clone Southern Blot

• A clone blot allows identification of the restriction fragments in cloned DNA that contain DNA sequences matching the probe DNA.
• Probe DNA in lane 1 extends across a restriction enzyme site, labeling two fragments.