How is it possible to produce transgenic organisms?
Recombinant DNA technology produces transgenic (genetically modified) organisms because:
The genetic code is universal - the same triplets code for the same amino acids in all organisms
Transcription and translation are also universal processes
Name the 6 stages to recombinant DNA technology
Isolating a gene
Amplification of a gene to produce many copies
Insertion of gene into a vector
Transformation of cells with the vector
Identification oftransformed cells
Culturing of cells which have taken up the vector
What are the three ways to isolate a gene?
Reverse transcriptase
Restriction endonucleases
Gene machine
Describe isolating a gene using reverse transcriptase
Find cells expressing the mRNA in large amounts and extract them
Mix the mRNAs with reverse transcriptase to convert them to DNA without introns
DNA polymerase is used to produce double-stranded copy of the gene
Describe isolating a gene using restriction endonucleases
REs are enzymes that cut DNA at palindromic recognition sites. Some REs leave sticky ends (exposed bases) and some leave blunt ends
One RE cuts at a recognition site at the start of the gene, and a different RE cuts at a recognition site at the end of the gene
This removes the double-stranded gene from the DNA
The introns are then removed from the gene
Describe isolating a gene using a gene machine
Contains a store of DNA nucleotides and a computer that can assemble them in a specific order
The gene's base sequence is typed in without introns, and the nucleotides are assembled in the correct order
DNA polymerase is used to produce a double-strabded copy of the gene
Describe stage 2
Done using polymerase chain reaction (PCR) which is an in vitro (outside cells) technique to make lots of copies of DNA
Outline the PCR process
DNA is heated to 95 degrees which breaks the hydrogen bonds between the bases, separating the strands
Then cooled to 55 degrees. This allows primers to anneal to the end of each strand
FreeDNA nucleotides are also attracted to their complementary bases
Then heated to 72 degrees, the optimum temperature for thermostable DNA polymerase to bind the primers and form phosphodiester bonds between nucleotides
The cycle is repeated to produce millions of copies of the original DNA
Describe the importance of primers
They allow DNA polymerase to bind to the end of each strand and start joining nucleotides
They're made to be complementary to the ends of strands so that only target DNA is amplified
They stop strands from joining back together
Describe a vector
A vector is a molecule that transfers a gene into the cell
Describe modifying the gene before stage 3
add promoter region to allow RNA polymerase to bind
add terminator gene to allow RNA polymerase to detach
Describe stage 3 - the insertion process
The gene and vector are cut with the same restriction endonucleases, leaving complementary sticky ends
DNA ligase forms phosphodiester bonds between nucleotides to splice the gene and plasmid together
Describe the 3 outcomes of insertion
A recombinant plasmid - target gene inserted
A non-recombinant plasmid - no target gene inserted
Twotarget genes stick together
Describe transforming cells with the vector
The plasmids and bacterial cells are mixed with calcium ions and exposed to heatshock
The cell surface membranes of the bacterial cells become more permeable, allowing plasmids to pass through the cytoplasm
Describe identification of the cells that have been transformed
Only 1% of bacteria are ever successfully transformed with a plasmid
Of the 1% of transformed cells, some won't have the recombinant plasmid
Marker genes:
A gene found on the plasmid that produces a protein which causes a visual effect
Marker genes can be found naturally on the plasmid or artificially inserted
Name stage 6
Cells with the recombinantplasmid are grown in large quantities