Molecular Biology: Recombinant DNA Technology

Molecular Biology Techniques

• Use enzymes to manipulate and investigate DNA.

Genes and DNA

• DNA is the genetic material.
• The ability to isolate and manipulate DNA allows for:
  • Isolation and modification of genes for function understanding.
  • Deriving gene sequences from mutant phenotypes.
  • Manufacturing proteins like insulin and factor VIII.
  • Creating vaccines.
  • Producing transgenic organisms and plants.
  • Diagnosis of genetic diseases.
  • Gene therapy development.

Recombinant DNA Technology (Gene Cloning)

• It is used in the isolation and manipulation of DNA.
• Achieved by isolating sections of DNA and creating multiple copies.
• The copies are known as Gene Cloning because clones are made of unique DNA pieces.

Molecular Cloning

• DNA pieces must be reproduced in large amounts for further study.
• Isolated DNA fragments are inserted into a cloning vector to create a recombinant DNA molecule.
• A cloning vector, like a plasmid, introduces DNA into a host organism and can self-replicate.
• The recombinant DNA molecule then replicates within a biological system to produce many copies.
• The identical copies are known as DNA or molecular clones.

Restriction Enzymes

• Restriction enzymes act as molecular scissors to cut DNA.
• They recognize specific nucleotide sequences, cutting both DNA strands at the sugar-phosphate backbone.
• First identified in bacteria as a defense against viral DNA by cutting it off.
• Different restriction enzymes recognize different nucleotide sequences forming a palindrome, which reads the same on both strands in the 5'-3' direction.
• Restriction enzymes cleave DNA, leaving cohesive (sticky) or blunt ends.

Restriction Enzyme Function

• Restriction enzymes cut genomes into numerous DNA fragments.
• EcoRI cuts the human genome into around 730,000 pieces.
• Restriction site frequency depends on the genome's GC or AT content.
• DNA sequence isn't random, so restriction sites can be close or far apart in different regions.
• Number and size of fragments depends on the restriction enzyme, genome size, and nucleotide abundance.
• 4-cutters are more common than 6-cutters.

Genomic Digest

• DNA is cut into different fragments during genomic digestion.
• Drosophila genomic DNA digested with EcoRI, and separated on agarose gel, appears as a smear.
• It's a smear because EcoRI cuts DNA into fragments of varying sizes, from tens to thousands of base pairs long.
• Each fragment is a restriction fragment.

Restriction Enzyme Mapping

• Restriction maps are created by cutting DNA with restriction enzymes.
• Gel electrophoresis separates the digested DNA by size.
• Fragment sizes are identified by comparing their movement in the gel to DNA fragments of known sizes.
• This determines how often each enzyme cuts and the position of their sites relative to each other.
• Different restriction enzymes are used in pairs to locate where their sites are relative to one another.
• Restriction enzyme maps of plasmids and genomes are necessary to locate genes and aid in their study and manipulation.

Recombinant DNA

• Large amounts of individual DNA fragments are required for further study and manipulation.
• Bacteria or yeast is used to replicate individual DNA fragments
• DNA must be introduced into a host organism through a mechanism.
• Plasmids, circular DNA molecules, serve as cloning vectors to transfer DNA fragments into a host bacterium.

Plasmids

• Plasmids allow replication independently of the bacterial chromosome because they contain an origin of replication.
• Plasmids are maintained in high copy numbers, ranging from 100 to 200, in each bacterium.
• They also contain antibiotic resistance genes that allow for selective growth of bacteria with plasmids.
• Plasmids used for DNA cloning have a Multiple Cloning Sequence (MCS).
• There are various types of plasmids, including cloning plasmids for cloning genes and expression plasmids for gene expression.

Cloning DNA Molecules

• A DNA molecule can be cloned by inserting it into a plasmid cloning vector.
• The plasmid and DNA fragment, cut with restriction enzymes create overhang sequences, pairing up using enzyme ligase.

Creating Recombinant DNA Molecules

• DNA cut by the same restriction enzyme has the same cohesive ends.
• Cohesive ends are complementary base pairs.
• Complementary base pairs anneal due to A-T and C-G hydrogen bonds.
• Free 3' OH and 5' PO4 groups are covalently joined by DNA ligase.

Cloning DNA

• To clone a DNA fragment, both the fragment and the cloning vector are cut by the same restriction enzyme.
• Both the vector and the fragments will have complementary cohesive ends.
• Cut DNA fragments and the cut vector are then mixed.
• DNA fragments anneal to cohesive ends in the vector
• DNA ligase ligates the DNA fragment and cloning vector to form a recombinant DNA molecule.

Cloned DNA Amplification in E. coli

• Individual recombinant plasmids are taken up by E. coli bacteria.
• Using the origin of replication (ori), recombinant plasmid is generated up to 100-200 copies in each bacterium.
• As bacteria divide, each contains 100-200 copies of the recombinant plasmid.

Plasmids for DNA Cloning

• Plasmids used for DNA cloning have individual restriction sites clustered together in a multiple cloning site where DNA inserts can be cloned.

Identifying Recombinant Plasmids

• In many cloning plasmid vectors, the MCS is within a lacZ gene.
• lacZ encodes β-galactosidase, which acts on X-gal and produces blue bacteria.
• The lacZ gene is disrupted when an insert is cloned.
• Plasmids with an insert no longer make β-galactosidase unable to cleave X-gal and produce white colonies.

Uses of Cloned DNA

• Clone and piece together genomic DNA for the mapping and sequencing of genes.
• Often occurs by creating a genomic DNA library.
• Identify genomic changes associated with specific phenotypes or diseases.
• Characterise genome organisation and location of repetitive DNA.
• Genetically engineer organisms by transferring genes between organisms.
• Express large amounts of protein from isolated genes, for use as antigens in vaccines or therapeutics.

Cloning Eukaryotic Genes

• It is often desired to express a eukaryotic protein for study or therapeutic purposes.
• Genomic DNA cannot be used because the presence of introns.
• The eukaryotic genome contains a large number of genes (20,000 - 50,000), and most have introns.
• Introns can be large (spanning 10-100kb), so mRNA must be isolated and reverse-transcribed 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.
• Harvesting mRNA from different cells allows for the discovery of which genes are expressed by which cells.

Complementary DNA (cDNA)

• Needed to express eukaryotic proteins or identify genes expressed in a particular cell type through DNA copies of transcribed mRNA.
• mRNA is produced when genes are transcribed.
• mRNA does not contain introns, but cloning RNA is not possible, only DNA.
• cDNA is a DNA copy of mRNA, produced using reverse transcriptase.
• cDNA libraries contain complementary DNA copies of the mRNAs in a cell population.
• The genes in a sampled population represent the extracted mRNA
• cDNAs are used in eukaryotic expression vectors to remove introns.

cDNA Libraries

• Each mRNA from a sample is cloned as a cDNA into a plasmid.
• A cDNA library consists of plasmids each with a single cDNA.
• Genes highly expressed in the tissue or cells where mRNA was isolated will be more frequent in the library.
• Those not expressed in the tissue will not be present in the library.
• Regulatory sequences and introns are absent in cDNA libraries.

Expression Plasmids

• Specialised plasmids are used to allow gene expression.
• Expression plasmids are cloning vectors designed to express cloned genes in bacteria, producing large quantities of the encoded protein.
• They have key bacterial sequences that direct transcription and translation of the cloned DNA.
• Eukaryotic expression plasmids are cloning vectors for expression of cloned genes in eukaryotic cells.
• They include key eukaryotic regulatory sequences that direct transcription and translation.

Nucleic Acid Hybridisation

• Used to identify nucleic acid fragments or clones with specific sequences.
• Relies on complementarity of nucleic acid strands, where complementary strands anneal.
• Used to identify DNA or RNA fragments with specific sequences
• The individual strands of DNA molecules are separated by heating or in alkaline conditions.
• Complementary strands will anneal to one another after cooling or neutralization - this is known as, "hybridize."
• Single DNA strands will also hybridise to complementary RNA molecules.
• ssDNA identifies complementary fragments of RNA or DNA

Nucleic Acid Hybridisation Technique

• Nucleic acid hybridization identifies DNA or RNA that matches a specific sequence.
• ssDNA can be used as a probe to identify DNA fragments or clones in a collection with a sequence complementary to the probe DNA.

DNA Probe Preparation

• DNA must be labelled before use as a probe for subsequent detection.
• The DNA probe is labelled by using DNA polymerase to incorporate labelled dNTPs.
• Labeled dNTPs can be made radioactive using 32P or by attaching a fluorescent or digoxygenin molecule.

Identifying DNA Fragments

• Nucleic acid hybridization identifies DNA fragments in the genome that contain specific genes.
• Southern hybridization (Southern blot), named after Ed Southern, is a technique used.
• Used to identify DNA fragments containing a specific gene or sequence after genomic DNA with a restriction enzyme.

Southern Blot

• DNA fragments are separated by gel electrophoresis then denatured in alkali.
• The DNA fragments are transferred to a DNA-binding membrane, such as nitrocellulose or nylon.
• A single-stranded, labelled DNA probe, made from a specific sequence of interest, is introduced in hybridization solution with the membrane.
• The labelled probe DNA hybridizes to the matching complementary DNA fragment on the membrane.
• The hybridize DNA can be identified in relation to being labelled by the probe.