Genetic engineering techniques
The ability to genetically engineer organisms is built on years of research and discovery on gene function and manipulation.
Important advances included the discovery of restriction enzymes, DNA ligases, and the development of polymerase chain reaction and sequencing.
First generation offspring are heterozygous, requiring them to be inbred to create the homozygous pattern necessary for stable inheritance.
Crop hybridization most likely first occurred when humans began growing genetically distinct individuals of related species in close proximity.
[3] In 1928 Frederick Griffith proved the existence of a "transforming principle" involved in inheritance, which was identified as DNA in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty.
Frederick Sanger developed a method for sequencing DNA in 1977, greatly increasing the genetic information available to researchers.
In 1970 Hamilton Smiths lab discovered restriction enzymes, enabling scientists to isolate genes from an organism's genome.
Polymerase chain reaction (PCR), developed by Kary Mullis in 1983, allowed small sections of DNA to be amplified (replicated) and aided identification and isolation of genetic material.
A simple screen involves randomly mutating DNA with chemicals or radiation and then selecting those that display the desired trait.
[15] The gene that provides resistance to the herbicide glyphosate was found after seven years of searching in bacteria living in the outflow pipe of a Monsanto RoundUp manufacturing facility.
Once isolated, additional genetic elements are added to the gene to allow it to be expressed in the host organism and to aid selection.
By mixing with phenol and/or chloroform, followed by centrifuging, the nucleic acids can be separated from this debris into an upper aqueous phase.
If the position of the gene can be determined using molecular markers then chromosome walking is one way to isolate the correct DNA fragment.
A selectable marker, which in most cases confers antibiotic resistance to the organism it is expressed in, is used to determine which cells are transformed with the new gene.
There are a number of techniques available for inserting the gene into the host genome and they vary depending on the type of organism targeted.
The only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation.
Chemical based methods uses natural or synthetic compounds to form particles that facilitate the transfer of genes into cells.
The gene is transfected into embryonic stem cells and then they are inserted into mouse blastocysts that are then implanted into foster mothers.
[48] Finding that a recombinant organism contains the inserted genes is not usually sufficient to ensure that they will be appropriately expressed in the intended tissues.
These include northern hybridisation, quantitative RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis.
Cre recombinase is an enzyme that removes DNA by homologous recombination between binding sequences known as Lox-P sites.
The breaks are subject to cellular DNA repair processes that can be exploited for targeted gene knock-out, correction or insertion at high frequencies.
[62] Recent advances have looked at combining multiple systems to exploit the best features of both (e.g. megaTAL that are a fusion of a TALE DNA binding domain and a meganuclease).
While a certain amount of off-target cleavage is acceptable for creating transgenic model organisms, they might not be optimal for all human gene therapy treatments.
[65] Access to the code governing the DNA recognition by transcription activator-like effectors (TALE) in 2009 opened the way to the development of a new class of efficient TAL-based gene editing tools.
Due to the presence of repeat sequences, they are difficult to construct through standard molecular biology procedure and rely on more complicated method of such as Golden gate cloning.
By pairing Cas proteins with a designed guide RNA CRISPR/Cas9 can be used to induce double-stranded breaks at specific points within DNA sequences.
CRISPR/Cpf1 is a more recently discovered system that requires a different guide RNA to create particular double-stranded breaks (leaves overhangs when cleaving the DNA) when compared to CRISPR/Cas9.
This method links a reverse transcriptase to an RNA-guided engineered nuclease that only makes single-strand cuts but no double-strand breaks.
It replaces the portion of DNA next to the cut by the successive action of nuclease and reverse transcriptase, introducing the desired change from an RNA template.
