Genomic and cDNA cloning Flashcards
What are cosmids?
A hybrid between lambda and a plasmid.
What are the benefits of using a cosmid?
It enables large pieces of DNA to be cloned (35kb).
How does the cosmid behave and function?
It uses the efficient infection of E.coli cells by lambda particles and once inside the E.coli behaves as a plasmid.
What is special about the lambda DNA part of this molecule?
Cohesive ends/cos sites that are 12 base complementary single stranded ends.
What is special about the cos sites?
The enzymes that package lambda DNA into the protein coat only require cos sites in order to function, and will package not only lambda DNA but any DNA that carries cos sites separated by 37-52kb of DNA.
What is an example of a cosmid?
pJB8. It is 5.4kb in size and has an ampicillin resistance gene, an ori site, a suitable cloning site and a small piece of lambda DNA containing the cos site.
What is the benefit of this cosmid?
Allow us to clone large pieces of DNA with the high efficiency of infection of lambda particles. The cos sites allow us to do that.
What happens when the cosmid infects the E.coli cell?
The DNA recircularises via the cos site. The infection is a highly efficient process.
How will the medium select for the E.coli cells that are infected with the cosmid?
Due to the ampicillin resistant gene, the E>coli cells containing the cosmid will be selected.
Will the cosmids gve rise to bacterial colonies or plaques?
Colonies.
What are cosmids used for?
Constructing genomic libraries.
What is a genomic library?
A collection of cloned fragments that encompass the whole genome of an organism. It will contain all the genes of the organism and the non-coding regions as well.
What is the equation for the number of clones needed?
N=ln(1-p)/ln(1-a/b) where N is the number of clones needed, P is the probability that the gene is present, a is the average size of DNA insert in vector and b is the total size of the genome.
What are problems with the construction of a genomic library?
6-cutter enzmes such as BamHI cuts on average once every 4096 nucleotides. Many fragments will be too small , and some too large to clone. Sau3A (4 cutter) will produce fragments that are too small to clone.
How can this problem with genomic libraries be solved?
Perform a partial digest with Sau3A (stop digestion going to completion and taking samples at various timepoints) and select sizes appropriate for cloning and clone into a BamHI-cut replacement vector.
What are all the steps in making a genomic library?
Partial digest with Sau3A, ligate into a BamHI cut cosmid, package into lambda particles and infect E.coli (with high efficiency) and plate onto agar and ampicllin and recover the colonies.
What are some methods to clone even larger fragments?
Bacterial artificial chromosomes (BACs) - based on F plasmid of E.coli and can clone fragments up to 300kb. Bacteriophage P1 that can clone up to 110kb and P1 derived artificial chromosomes (PACs) that are cosmid-like vectors based on P1 and can clone up to 300kb.
What is cDNA cloning?
Copy or complementary cloning of DNA copies of eukaryotic mRNA.
Why would cDNA cloning be done?
As eukaryotic genomes are large and most is non-coding, so this can allow cloning of coding regions alone without introns. It takes advantage of differential gene expression.
How can mRNA be isolated?
Isolate the total RNA from the tissue of interest and purify it (using PolyA-RNA binding by oligo-dt chromatography) and use this as the substrate for cDNA snythesis.
How can the mRNA be made into a double strand?
Using reverse transcriptase to synthesise a complementary DNA strand to the RNA template.
Why does the mRNA need to be made into a double strand?
Restriction enzymes can only work on double stranded templates.
Why do oligo(dT) primers need to be bound to the mRNA?
They can bind to the Poly(A) tails and act as a primer for the reverse transcriptase.
What molecule is formed after reverse transcriptase has acted?
A DNA-RNA hybrid double stranded molecule.
