21.1 PRODUCING DNA FRAGMENTS
Wednesday, 16 February 2022
15:02
Recombinant DNA is when certain genes or fragments from one organism is mixed
with the DNA of another organism.
The resulting organism is known as a genetically modified organism (GMO).
There are many stages to making proteins using DNA technology of gene transfer
and cloning:
● Isolation of DNA fragments which code for the desired proteins.
● Insertion of DNA fragment into vector.
● Transformation, transferring the vector with DNA into a suitable host cell
(bacteria).
● Identification of host cells which have successfully taken up the gene by the
use of gene markers.
● Growth/cloning of the population of successful host cells.
There are 3 main methods to producing DNA fragments to isolate desired genes:
● Using reverse transcriptase to convert mRNA to complimentary DNA (cDNA).
● Using restriction endonuclease enzymes to cut a fragment containing the
desired gene for DNA.
● Creating the gene in a gene machine.
Reverse transcriptase forms DNA from mRNA.
Most cells only contain 2 copies of each gene, however they can contain many mRNA
molecules complimentary to the target gene.
● Easier to obtain the mRNA than directly obtaining the fragment of DNA.
A cell which readily produces the desired protein is selected and they have large
quantities of the relevant mRNA to be isolated and extracted.
● Reverse transcriptase and free nucleotides are used to make the cDNA from
the mRNA.
● DNA polymerase and free nucleotides form the other strand of DNA on the
cDNA template.
● This double strand of DNA is the required DNA fragment.
Restriction endonucleases are enzymes which cut DNA at specific base sequences,
recognition sites.
● Can be used to cut out a desired gene from the rest of the genome.
Restriction endonucleases can cut between 2 opposite base pairs, leaving blunt ends.
, They can also cut in a staggered fashion, leaving each strand of the DNA having
exposed sticky ends.
● Due to complimentary base pairing, the sticky end will pair with another piece
of DNA with a sticky end which has been cut from the same restriction
endonuclease.
● As they pair, H bonds form between bases and DNA ligase forms the
phosphodiester bonds between the nucleotides.
It is possible to manufacture genes in a lab using a gene machine.
The fragments of DNA can be made from scratch without the need of a DNA
template as a database contains the information needed to produce the DNA
fragment.
● The desired sequence of bases is determined from the desired protein by
determining the amino acid sequence.
● Desired sequence of nucleotide bases for the gene is fed into the computer.
● Sequence checked for biosafety and biosecurity to ensure it reaches
international standards and ethical requirements.
● Computer designs a series of oligonucleotides, small overlapping single
strands of nucleotides.
● The oligonucleotides are formed by adding one nucleotide at a time in the
required sequence.
● The oligonucleotides join together to make the gene.
Wednesday, 16 February 2022
15:02
Recombinant DNA is when certain genes or fragments from one organism is mixed
with the DNA of another organism.
The resulting organism is known as a genetically modified organism (GMO).
There are many stages to making proteins using DNA technology of gene transfer
and cloning:
● Isolation of DNA fragments which code for the desired proteins.
● Insertion of DNA fragment into vector.
● Transformation, transferring the vector with DNA into a suitable host cell
(bacteria).
● Identification of host cells which have successfully taken up the gene by the
use of gene markers.
● Growth/cloning of the population of successful host cells.
There are 3 main methods to producing DNA fragments to isolate desired genes:
● Using reverse transcriptase to convert mRNA to complimentary DNA (cDNA).
● Using restriction endonuclease enzymes to cut a fragment containing the
desired gene for DNA.
● Creating the gene in a gene machine.
Reverse transcriptase forms DNA from mRNA.
Most cells only contain 2 copies of each gene, however they can contain many mRNA
molecules complimentary to the target gene.
● Easier to obtain the mRNA than directly obtaining the fragment of DNA.
A cell which readily produces the desired protein is selected and they have large
quantities of the relevant mRNA to be isolated and extracted.
● Reverse transcriptase and free nucleotides are used to make the cDNA from
the mRNA.
● DNA polymerase and free nucleotides form the other strand of DNA on the
cDNA template.
● This double strand of DNA is the required DNA fragment.
Restriction endonucleases are enzymes which cut DNA at specific base sequences,
recognition sites.
● Can be used to cut out a desired gene from the rest of the genome.
Restriction endonucleases can cut between 2 opposite base pairs, leaving blunt ends.
, They can also cut in a staggered fashion, leaving each strand of the DNA having
exposed sticky ends.
● Due to complimentary base pairing, the sticky end will pair with another piece
of DNA with a sticky end which has been cut from the same restriction
endonuclease.
● As they pair, H bonds form between bases and DNA ligase forms the
phosphodiester bonds between the nucleotides.
It is possible to manufacture genes in a lab using a gene machine.
The fragments of DNA can be made from scratch without the need of a DNA
template as a database contains the information needed to produce the DNA
fragment.
● The desired sequence of bases is determined from the desired protein by
determining the amino acid sequence.
● Desired sequence of nucleotide bases for the gene is fed into the computer.
● Sequence checked for biosafety and biosecurity to ensure it reaches
international standards and ethical requirements.
● Computer designs a series of oligonucleotides, small overlapping single
strands of nucleotides.
● The oligonucleotides are formed by adding one nucleotide at a time in the
required sequence.
● The oligonucleotides join together to make the gene.
