Genetic Engineering Flashcards
What does gene cloning involve?
The amplification of DNA or RNA with the aim of obtaining large quantities of defined pieces of DNA.
What falls under genetic engineering?
Gene cloning, expression
DNA sequencing
Protein engineering
What are the two types of cellular cloning?
Reproductive cloning; which requires nuclear transfer into a enucleated oocyte.
Therapeutic cloning; in which embryonic or adult stem cells are used for therapy.
What are the two ways to clone?
Cloning vectors
In vitro cloning via PCR
Target materials is mRNA and genomic library.
What are genomic libraries?
A pool of recombinant molecules such as YACs, bacteriophage, BACs or plasmids that have inserts (usually overlapping) that cover the entire genome.
How do you map genes?
The locus needs to be found in the library - called screening the library. This can be done using known sequences as probes. (Associated sequences or from other species). Clones carrying the region of interest are identified and may be overlapping in the DNA they contain.
DNA can be cut from these vectors eg start with YAC vectors then placed into smaller vectors for manipulations i.e to sequence the DNA.
Several YAC libraries have been produced covering the human genome for this purpose. The smaller vectors allow sequencing of the DNA inserts these are also lined up to form the full sequence of the gene and it’s surrounding DNA.
How are DNA fragments ligased?
The DNA fragments and the linearised vector molecules are mixed in a tube. The sticky ends with the identical overlap anneal with each other.
T4 DNA ligase is added which forms a phosphodiester bond between the 3’-OH and 5’P-groups of the sticky ends.
Formation of recombinant molecules (vector plus fragment) are formed.
What is transformation?
The assisted uptake of naked DNA by a host cell.
This means the recombinant molecules are transferred into a living cell called a host cell.
This uptake is mediated by making cells competent.
Hosts can either be bacterial (E. coli) or eukaryotic ie. yeast (S. cerevisiae).
These cells are called transformants and these form the so-called gene library.
How did cloning cDNA come about?
Pioneered by cloning beta-globin gene.
Give three facts about cDNA libraries.
Libraries should have clones that have all mRNA.
cDNA libraries from different tissues carry different mRNA expression therefore libraries can be constructed for each tissue.
cDNA cloning will not give information about promoters, enhancers and transcription binding sites.
What are the advantages of genomic DNA?
Cloning of non-expressed genes is possible
Cloning of intergenic DNA
Cloning of intronic DNA
Cloning of regulatory elements of genes (upstream/downstream elements).
What are the disadvantages?
Library contains lots of non-coding DNA.
Large insert vectors are required (or a very high clone number) to obtain satisfactory gene representation.
Genomic clones are not expressed in bacteria or fungi because there is no splicing mechanism.
What are the advantages of cDNA cloning?
Library contains only genes from expressed genes.
Clones are readily expressed in bacteria because there are no intronic sequences in cDNA.
What are the disadvantages of cDNA cloning?
Library does not contain any DNA which is not showing up in mRNA.
Low abundancy transcripts are difficult to obtain (probably including many relevant genes).
What are the applications of genetic engineering?
Drug development Bio markers Animal models of human diseases Forensic sciences GM crops and transgenic plants.
