Mod 10- Genetic Engineering Flashcards
What is molecular cloning?
Cutting, joining, and propagating recombinant DNA
It involves isolating DNA fragments and allows for large-scale production and analysis.
What are the two types of gene libraries mentioned?
- Genomic
- cDNA (derived from mRNA)
Genomic libraries contain all genes, while cDNA libraries contain only expressed genes.
What are the steps involved in molecular cloning?
- Isolate DNA
- Cut DNA
- Join to a vector
- Introduce recombinant vector into bacteria
- Amplify recombinant DNA by bacterial growth
These steps enable the production of recombinant DNA.
What is the function of restriction endonucleases?
Cuts palindromic sequences and creates ‘sticky ends’ that can be rejoined
Different restriction endonucleases have different sequence specificities.
What is the purpose of using a phosphatase in cloning?
Prevents recircularization of plasmid
This ensures that only the recombinant plasmid is propagated.
What is a cDNA library?
- Comprises expressed genes (transcriptome) from mRNA
- ‘Libraries’ from different tissues contain different sequences
- No untranscribed sequences
What is a genomic library?
- Comprises sequences representing the genome = same in all tissues
- Includes introns and regulatory sequences as well as exons
- “Raw material” for gene mapping and genome projects
Why is genetic engineering used for therapeutic proteins?
Many proteins have therapeutic value but are hard to obtain
The process allows for easier production and purification of these proteins.
What are the advantages of using bacteria as host systems in protein production?
- Cheap
- Fast growing
- Easy to maintain
- Stable
Bacteria are often preferred for their efficiency in producing proteins.
What are the disadvantages of using bacteria for protein production?
- Proteins may be insoluble/denatured
- No post-translational modification (e.g. methylation, acetylation etc)
These factors can affect the functionality of the produced proteins.
What are the advantages of using animal cells in protein production?
- Post-translational modification
- Soluble/properly folded
These characteristics are important for the functionality of therapeutic proteins.
What is the disadvantage of using animal cells for protein production?
Expensive and unstable (may be rejected)
What was the case study discussed regarding insulin?
Producing human insulin in bacteria
By processing insulin and synthesising the 2 chains apart then joining them to form the folded structure
This method avoids immunological problems and the possibility of cross-species infection associated with animal sources.
What is required to produce recombinant proteins?
- Fuse coding region of gene to a strong promoter
- Insert recombinant gene into host
- Host multiplies and overproduces protein
- Purify protein
This general principle applies to many therapeutic proteins.
What are the intended learning objectives for this lecture?
- Basic principles of molecular cloning
- Use of recombinant DNA techniques to create gene libraries
- Differences in composition of genomic and cDNA libraries
- Employment of techniques to produce therapeutic proteins
These objectives guide the learning process for students.
