2.Production of a recombinant product
Monday, February 26, 2024 1:05 PM
Recombinant DNA Technology
Definitions: Recombinant DNA Technology involves combining DNA from different
species to create new genetic combinations.
Applications:
• producing DNA,
• proteins,
• biochemical pathway products
• chemical structures for science
• medicine,
• Agriculture
• industry.
Isolating Gene of Interest WHATS NEEDED TO CLONE GOI:
• Source DNA containing the gene of interest can be isolated from bacteria, yeast, • GOI
fungi, plant cells, or animal cells. • Restriction enzymes
• Vector to receive
• Genomic DNA extraction involves cell culture growth, cell lysis, removal of • Mechanism to "glue" GOI into vector
contaminants, and DNA concentration.
• Polymerase Chain Reaction (PCR) is used to separate the gene of interest from
chromosomal DNA by amplifying a specific DNA sequence.
• PCR involves denaturation, annealing, and extension steps to replicate DNA in a
test tube.
Production of Recombinant Product
Cloning process:
1. cloning the Gene of Interest (GOI) into a vector using restriction enzymes and a
mechanism to insert the GOI.
2. Restriction enzymes cut DNA at specific nucleotide sequences, creating sticky or
blunt ends for ligation.
3. Vectors, like plasmids, are circular DNA molecules used to transport foreign genes
into host cells for replication and expression.
4. Plasmids must have an origin of replication, selectable markers, and restriction
enzyme sites to be useful for recombination technology.
Characteristics of Vectors :
• must be circular, large enough to hold the gene of interest, and contain an Origin of
DNA replication (ORI).
• Multi-cloning sites (MCS) provide multiple restriction enzyme sites for gene
insertion.
• Marker genes and control sequences like transcription promoters help identify cells
with the vector and ensure gene expression.
• Copy number, the quantity of plasmids per bacterial cell, is crucial for successful
cloning, ranging from 1 to hundreds per cell.
Plasmid Copy Number and Cloning Vectors
DNA Ligase:
• repairs DNA breaks by joining nucleotides in a DNA strand.
• complements restriction enzymes by linking DNA fragments covalently.
• Enzyme acts as a 'spot welder' forming covalent bonds between DNA strands.
Cloning Technique with DNA Ligase
• Enzymes with staggered cuts create complementary ends for ligation.
• Sticky ends must be complementary for successful ligation
Bacterial Transformation and Recombinant Product Production
Bacterial Transformation:
• Vectors need to be incorporated into bacterial cells for replication or expression.
• Transformed cells are selected using antibiotic resistance genes.
Transformation Methods:
1. electroporation
2.CaCl2 (calcium chloride)/Heatshock
Recombinant Product Production:
• Verification steps include plasmid isolation, restriction digestion, PCR, and
sequencing.
• Expression systems like bacteria, fungi, yeast, or mammalian cells are used.
• Intracellular and extracellular product expression methods differ in purification
ease.
Cell Line Development
Cell Line Construction
expressing proteins in :
Monday, February 26, 2024 1:05 PM
Recombinant DNA Technology
Definitions: Recombinant DNA Technology involves combining DNA from different
species to create new genetic combinations.
Applications:
• producing DNA,
• proteins,
• biochemical pathway products
• chemical structures for science
• medicine,
• Agriculture
• industry.
Isolating Gene of Interest WHATS NEEDED TO CLONE GOI:
• Source DNA containing the gene of interest can be isolated from bacteria, yeast, • GOI
fungi, plant cells, or animal cells. • Restriction enzymes
• Vector to receive
• Genomic DNA extraction involves cell culture growth, cell lysis, removal of • Mechanism to "glue" GOI into vector
contaminants, and DNA concentration.
• Polymerase Chain Reaction (PCR) is used to separate the gene of interest from
chromosomal DNA by amplifying a specific DNA sequence.
• PCR involves denaturation, annealing, and extension steps to replicate DNA in a
test tube.
Production of Recombinant Product
Cloning process:
1. cloning the Gene of Interest (GOI) into a vector using restriction enzymes and a
mechanism to insert the GOI.
2. Restriction enzymes cut DNA at specific nucleotide sequences, creating sticky or
blunt ends for ligation.
3. Vectors, like plasmids, are circular DNA molecules used to transport foreign genes
into host cells for replication and expression.
4. Plasmids must have an origin of replication, selectable markers, and restriction
enzyme sites to be useful for recombination technology.
Characteristics of Vectors :
• must be circular, large enough to hold the gene of interest, and contain an Origin of
DNA replication (ORI).
• Multi-cloning sites (MCS) provide multiple restriction enzyme sites for gene
insertion.
• Marker genes and control sequences like transcription promoters help identify cells
with the vector and ensure gene expression.
• Copy number, the quantity of plasmids per bacterial cell, is crucial for successful
cloning, ranging from 1 to hundreds per cell.
Plasmid Copy Number and Cloning Vectors
DNA Ligase:
• repairs DNA breaks by joining nucleotides in a DNA strand.
• complements restriction enzymes by linking DNA fragments covalently.
• Enzyme acts as a 'spot welder' forming covalent bonds between DNA strands.
Cloning Technique with DNA Ligase
• Enzymes with staggered cuts create complementary ends for ligation.
• Sticky ends must be complementary for successful ligation
Bacterial Transformation and Recombinant Product Production
Bacterial Transformation:
• Vectors need to be incorporated into bacterial cells for replication or expression.
• Transformed cells are selected using antibiotic resistance genes.
Transformation Methods:
1. electroporation
2.CaCl2 (calcium chloride)/Heatshock
Recombinant Product Production:
• Verification steps include plasmid isolation, restriction digestion, PCR, and
sequencing.
• Expression systems like bacteria, fungi, yeast, or mammalian cells are used.
• Intracellular and extracellular product expression methods differ in purification
ease.
Cell Line Development
Cell Line Construction
expressing proteins in :
