cDNA & Clone Collections on ZAGENO
A genome - the entire genetic material present in an organism - can be anywhere between 1.8 kb to 130 Gb base pairs long. So when studying the genetics of an organism, scientists like to break down their chosen genome of interest into manageable pieces. Once done, it is then possible to test and determine the DNA segment’s function.
To begin such experiments, clone collections are often used. Put simply; these are banks of genetically modified, easily transfected microbial communities. Individual microbes hold a segment of the target genome, and together, the microbial community collectively holds the entire genome. So a clone collection is an extensive ‘library’ of cDNA or genome clones from an organism of interest, like human, mouse, or rat, in select expression vectors.
In building such clone collections, scientists utilize natural occurring processes to simulate DNA recombination. That is, in cloning, DNA is fragmented into smaller segments of whole genes, promoter sequences, non-coding sequences or random fragments via restriction enzymes. These are then ligated into desired DNA strands of target genes or sequences, forming recombinant DNA. Once the recombinant DNA is ready, it is inserted into a vector which is then transfected into a competent cell. Scientists can then screen colonies for those that were successfully transfected, usually with a white-blue test.
cDNA collections only contain active genes . Since cDNA is made from a reverse transcriptase enzyme, which converts mRNA back into DNA, the collection will contain only the genes which were transcribed by DNA.
A clone collection is a large source of genetic material of a target organism, so scientists may perform more accurate tests, ranging from genetic fingerprinting to protein production.
Clone collections usually come in a variety of clone vectors, each with a range of insert sizes. For example, a plasmid has space to insert up to 15k base pairs, whereas Yeast Artificial Chromosomes (YACs) range from 250 to 2000k bp. Which vector is right for you depends on the size of the genome of your target organism.
Plasmid: a double-stranded circular DNA, independent from chromosomal DNA, and is found in most bacteria. Plasmids range from 2 – 4 kb in length and can carry inserts up to 15kb.
Phage lambda: a strand of viral DNA that infects E. coli. This virus is 48.5kb in length and can carry inserts up to 25kb. These inserts are joined with the viral DNA, which codes for the construction of viral particles. These then infect the host cell and then reformed with the help of local DNA ligase.
Cosmid: a hybrid of a plasmid vector and the Phage lambda. More specifically, a cosmid is a plasmid that contains the cos sequence from the Phage Lambda virus as described previously. This allows the cosmid to pack itself into viral particles, that once transduced into a host cell, the cosmid reassembles itself into a plasmid with the help of the DNA ligase. This mechanism allows cosmids to hold up to 45kb of inserts.
Bacteriophage P1 Vector: a linear DNA strand that can maintain inserts up to 70 – 100 kb in size. Similar to phage lambda and cosmids, Bacteriophage P1 vector DNA can package itself into particles and infects a host cell where the p1 vector then circularizes itself.
Bacterial Artificial Chromosome: BACs are circular DNA that is around 7 kb in length, capable of holding inserts up to 300 kb in size, and contain the crucial Fertility factor. This F-factor allows for the even distribution of plasmids after cell division, thus ensuring the transfer of your DNA insert.
Yeast Artificial Chromosome: YACs are linear DNA molecules of a normal yeast chromosome that can have inserts around 250 – 2000 kb in size.