Molecular Biology: PCR and DNA Cloning

• Molecular biology is applicable to medicine, with applications such as gene therapy and disease analysis.
• Molecular biology developments have been recognized with Nobel Prizes for tools such as restriction enzymes and genome editing.
• Molecular biology improves human health.
• Molecular cloning involves recombinant DNA technology and is used to preserve and amplify DNA sequences.
• Applications of molecular cloning include protein gene product production (recombinant expression) and mRNA vaccine production.

Obtaining DNA of Interest

• PCR, polymerase chain reaction
• It is an amplification of DNA from very small amounts
• It requires thermophilic DNA polymerase, which doesn't denature at high temperatures.
• Three steps are required 1. Denature, 2. Anneal, 3. Extend and must be repeated.
• Amplifies specific regions
• It is a fast process at under 2 hours
• Sensitive
• Cloning, genetic testing, detecting virus infections and preimplantation genetic diagnosis are possible applications.

Reverse Transcriptase PCR (RT-PCR)

• Used to obtain DNA from RNA using reverse transcriptase.
• It is used to obtain transcriptome (collection of all transcribed genes in a particular cell, gene expression pattern/program)
• Used to compare gene expression programs (transcriptomes) from different cells.

RT-PCR Protocol

• RNA is the starting template
• Single stranded (ss) complementary DNA is made from RNA template
• PCR amplifies cDNA to be used with enzymes to amplify cDNA of a single gene or all genes transcribed in a cell (transcriptome)
• Restriction endonucleases manipulate DNA for cloning and cleave double-stranded DNA

Restriction Endonucleases

• Bacterial enzymes are used to restrict foreign infections
• Digests within a double stranded DNA sequence (endonuclease exonucleases digest nucleic acid termini)
• Specific sequence recognition by enzymes
• This can produce cohesive or blunt DNA ends if from Escherichia coli

Restriction Enzymes

• Recognize palindromic sequences, with the same sequence when read 5' to 3' on both DNA strands.
• Molecular cloning involves amplified DNA using PCR and digested DNA using restriction enzymes.
• Gel electrophoresis separates and isolates desired DNA.
• Gel electrophoresis separates and visualizes DNA, RNA and proteins.
• Molecules separate based on size, shape and charge.
• Linear DNA separates by size
• DNA staining is done with fluorescent dyes.
• It appears with fluorescence upon UV light exposure
• DNA can then be cut out and purified for further use
• It can also be labeled and detected with covalently linked fluorophores or radioisotopes.
• Plasmid Vectors are needed.

Plasmid or Vector

• Double stranded, circular DNA.
• Is a vehicle to carry DNA into cells.
• Functions to amplify and isolate lots of DNA and is can be utilized for gene expression.
• Plasmid DNA contains the multiple cloning site (MCS); multiple sites for restriction enzymes and the Origin of replication to autonomously replicate in host
• Ligation and transformation are needed to place target DNA to a plasmid
• Ligates: Covalently link the two DNA pieces
• Transform: Introduce into bacteria (phenotype of bacteria is transformed)
• To make recombined DNA digest, ligate and transform.

Recombinant DNA

• Ligase is used in production of recombinant DNA molecules.
• It can use identical overhangs the Watson-Crick base pairing.
• A new phosphodiester bonds that are double stranded is created.
• Recombined DNA can be Heterologous.
• Heterologous expression is gene expression in a host cell that doesn't express that gene.
• Examples include recombinant insulin production and SARS-CoV2 nucleic acid vaccines.

Recombinant Insulin

• Transcription recombinant genes that is driven by a promoter
• mRNA is translated by host cell, and recombinant protein produced
• Purification (separation from bacterial proteins) and manipulation required)
• This occurs from heterologous expression with expression of a gene product in cells that do not express the gene
• It is an easier and cheaper process with large yields.
• There are differences between bacterial vs eukaryotic expression of recombinant proteins.
• Post-translational modifications, disulfide bond formation

Blotting and Hybridization

• Used to detect specific DNA, RNA or protein, amongst a mixture of different molecules, using the following approaches:
• Southern blotting; detect quantitate specific genes (DNA)
• Northern blotting; measure size, quantitate mRNA
• Western blotting; detect post-translational modifications, quantitate protein Southwestern blotting; detects DNA-binding proteins; proteins blotted (western) then probed with DNA (Southern)
• DNA probes with sequence complementarity target DNA for southern and northern blots
• Western blotting uses antibody detection, and has a protocol involving the following:
• Gel electrophoresis; molecules are separated with DNA being digested prior to protein loading.
• Transfer contents of gel to paper preserving the separating pattern.
• Afterwards single stranded DNA is added and will base pair to target sequence. Antibody is also applied with the target that is paired with a fluorescent probe
• Afterwards visualize the blot depending what DNA, enzyme, antibody, or fluorescence used.
• Hybridization occurs through base pairing - DNA/DNA, RNA/DNA, or RNA/RNA
• DNA microarrays allow high throughput analysis.
• The expression levels of 12,000 genes; brightness/color correlates to change in the expression of a particular gene.
• Microarrays are a simultaneous analysis that have the following factors:
• Each spot is unique (contains one gene or sequence from one gene)
• The precise location and sequence of each spot in a computer database can be located.
• Utilize hybridization, complementary base-pairing probes to match sample DNA to microarray library DNA.
• Ex: Amplichip, used to assess personalized drug dosages
• Gene expression profiling analyzes microarray protocols for a high-throughput comparison of gene expression between normal and tumor cells.
• mRNA is isolated (via RT-PCR) and is the transcriptome or gene expression program
• Fluorescently labeled cDNA is applied
• Hybridization of cDNA washes sequences with the ID of the well being known, allowing for expression to be analysed.

Gene Analysis

• A gene has various factors. These include:
  • Each column: Different tumor sample
  • Each row: Different gene
  • The relative expression of a single gene from a single tumor is indicated in the fluorescence in each well
  • Comparing of wells shows expression differences
  • Tumors can be classified into distinct classes based on gene expression patterns.
• ELISA (Enzyme-Linked Immunosorbent Assay) can quantitate protein analytes (peptides, antibodies, hormones, etc.)
• It can measure antibody in patient blood.
• Has various factors. These include:
• 1. Immobilized antigen
• 2. Primary antibody
• 3. Secondary antibody
• 4. Enzyme colorimetric assay or fluorescence label proportional to 1º Antibody or antigen
• Examples are ELISA diagnostic test for HIV.
• It is also applicable in vaccines