PCR and Recombinant Protein Production Quiz

Basic Biotechnology Techniques

• Polymerase Chain Reaction (PCR) is an in vitro technique that amplifies specific DNA sequences.
• PCR uses DNA polymerase to amplify small amounts of DNA for further testing.
• PCR products are substrates for the next reaction cycles.
• PCR is a transformation of a set of substances.
• Kary Mullys received the Nobel Prize in Chemistry in 1993 for PCR.

PCR Procedure

• PCR involves three main steps: denaturation, annealing, and extension.
• Denaturation (94°C): Separates the DNA double helix into two single strands.
• Annealing (57°C): Primers bind to their complementary sequences on the single strands.
• Extension (72°C): DNA polymerase extends the primers using nucleotides, creating new DNA strands. It's repeated approximately 30 times.
• The number of DNA copies doubles in each cycle (e.g., 2³⁰ copies)
• DNA template with a sequence of interest
• Primers
• dNTPs
• Polymerase
• Mg2+
• The cycle (denaturation, annealing and elongation) is repeated approximately 30 times, doubling the amount of target DNA each time.

PCR Essentials

• DNA Sample
• dNTPs (dATP, dGTP, dCTP, dTTP)
• Forward primer
• Reverse primer
• Enzyme (Taq Polymerase)
• Mg2+

PCR Principles

• PCR takes a specific DNA sequence and amplifies it
• DNA polymerase is used in the process
• One reaction product becomes the next reaction's substrate
• Transformation of a set of substances

Primer Design

• Primer length is typically 15-30 nucleotides.
• Primer G+C content is 40-60%.
• The melting temperatures (Tm) of forward and reverse primers should not vary significantly.
• Avoid repetitive or complementary sequences to prevent primer dimers or unproductive PCR.
• Repetitive sequences can lead to primer dimers or unproductive PCR.
• Avoiding these types of sequences helps ensure PCR efficiency and specificity.

Cloning

• Gene Cloning is the process of isolating and copying a gene of interest from an organism's DNA.
• Creating many copies of a gene of interest.
• Using a plasmid vector to insert the gene.
• The resultant protein expression using gene cloning depends on cellular conditions.

Genetic Tools - Enzymes

• Methylases: Modify DNA.
• Endonucleases: Cut DNA.
• Ligases: Paste DNA.
• Quinasies: Phosphate addition
• Phosfatases: Phosphate elimination
• Polymerases: Synthesis of DNA
• Exonucleases: Degrade DNA

General Cloning Protocol

• Cut DNA fragments using restriction enzymes.
• Anneal (join) the DNA fragment to the plasmid.
• Seal the gaps in the plasmid (join) using DNA ligase to make the plasmid.
• The resulting recombinant plasmid containing the gene of interest is introduced into a host cell (e.g., bacteria).
• These cells replicate the plasmid, thereby producing multiple copies of the desired gene.

Cloning Vector Elements

• Antibiotic resistance (AR) gene allows selection of successful clones.
• Multiple Cloning Site (MCS) contains unique restriction enzyme cutting sites to facilitate insertion.
• Ori (Origin of replication) regulates plasmid replication inside host cell.
• pUC19 is a common Cloning Vector which contain AR, MCS, and Ori

Cloning Vector Elements - Additional notes

• Vectors need necessary nucleic acid sequences for replication in the host cell.
• The sequence (ori) initiates replication.
• A sequence rich in A and T but poor in G and C in the replication origin allows for easy DNA strand separation.
• Important regulatory and initiation proteins.
• DNA polymerase for replication

Expression Vectors

• Used for high-level production of proteins of interest.
• Contain a strong promoter for efficient gene transcription.
• Include a ribosome binding site (RBS) for efficient translation initiation.
• May include a selectable marker to identify cells carrying the expression vector.
• A termination region.
• A polyadenylation signal.
• pGEX-123 is an example of an expression vector.

Protein Overexpression

• Bioproduction of recombinant protein in a certain amount for various purposes (research, biomedical, pharmaceutical, etc.)
• Maintains native protein properties

Protein Overexpression Methods

• In vitro: Transcription and translation using cell extracts
• In vivo: Introduces DNA coding sequences into a cell lineage

Protein Purification

• Multi-step process for separating protein of interest from a mixture of proteins.
• Separation techniques depend upon protein properties
• General scheme: Protein release, contaminant removal, selective separation, characterization.

Protein Purification Techniques

• Different methods based on solubility, electric charge, polarity, size/mass, and binding specificity are used.
• Techniques like precipitation, chromatography, electrophoresis, and affinity chromatography are common.

Recombinant Protein

• Recombinant proteins are created by inserting a gene of interest into a suitable vector.
• Subsequently, the expression of the inserted gene yields protein products in the host cell in large quantities.

Protein Quality Control

• Quality control is important to ensure research integrity
• Wasteful resource use.
• Wrong conclusions should be avoided

Factors Affecting Recombinant Protein Expression

• Promoter efficiency
• Translation starting sequences
• Codon usage
• mRNA structure
• Plasmid copy number
• Plasmid stability
• Host cell physiology

Protein Characterization

• Used to determine physical, chemical, and biological properties of a purified protein.
• Methods include absorbance, fluorescence, circular dichroism (CD), dynamic light scattering (DLS), and differential scanning calorimetry (DSC).

Organisms for Expression Vectors

• Bacteria
• Yeasts
• Insect cells
• Mammalian cells
• Plants
• Animals
• Baculovirus

Protein Expression in Yeast (Pichia pastoris)

• Pichia pastoris is a methylotrophic yeast, utilizing methanol as a carbon source when glucose is absent.
• The AOX1 promoter is used.

Protein Expression in Yeast (Pichia pastoris) - continued

• AOX1 has low specific activity, but it's expressed in substantial amounts.
• In the presence of large amounts of methanol.
• Alcohol Oxidase is a critical enzyme in this process.

Protein Expression in Yeast (Pichia pastoris) - continued

• Important for protein processing, folding, and post-translational modifications
• Some vectors allow cloning of genes near the AOX1 promoter allowing easy construction.
• A promoter inactive in the presence of glucose, but inducible in the presence of methanol offers a controlled environment for protein production.

Baculovirus as Expression System

• Baculoviruses are insect viruses that can be used to express proteins in insect cells.
• Recombinant viruses are formed by introducing a gene into a suitable virus vector.
• The main advantage is virus multiplication within host cells without causing occlusion bodies.

Plants as Expression Systems

• Agrobacterium tumefaciens is a soil bacterium.
• The plasmid Ti from A. tumefaciens is used to transform plants.
• The T-DNA from Agrobacterium contains the gene of interest, integrated into the plant's genome, which is then further processed. Methods for further processing vary based on the specific desired product.

Transgenic Animals

• Transgenic animals with a gene of interest inserted into their genome are used for expression systems.
• Several methods exist and procedures vary based on desired outcome.

PCR in detail

• Basic process detailed in a diagram.
• Separate steps and requirements for PCR process including the use of enzymes and specific temperatures required.
• Explain in clear terms the importance of each step and how the process works.

Test your knowledge on the principles of PCR and recombinant protein production. This quiz covers essential concepts such as the importance of PCR enzymes, the DNA replication process, and the role of vectors in biotechnology. Perfect for students of biotechnology or anyone interested in genetic engineering.