GENE TECHNOLOGY Flashcards
(53 cards)
What is a genome
the entire set of DNA, including all the genes in an organism
what parts of DNA does gene sequencing work on
fragments of DNA
what is a proteome of an organism
all of the proteins that are made by it
why is it easy to determine the proteome of simple organisms
eg a bacteria
doesn’t have much non coding DNA
therefore relatively easy to determine the proteome from the DNA sequence
why is it hard to determine the proteome of a complex organism
Contain larger sections of non coding DNA
Also have regulatory genes which determine when some genes coding for some proteins are switched on and off
Makes it hard to translate the genome to proteome
what does recombinant DNA technology involve
transferring a fragment of DNA from one organism to another
What are the 3 ways DNA fragments can be made
Reverse transcriptase
Restriction endonuclease enzymes
Using a gene machine
How do you make DNA fragments using reverse transcriptase
mRNA complementary to the gene and easier to obtain as more molecules
It is obtained by isolating it from cells, its then mixed with free DNA nucleotides
mRNA used as template to make lots of DNA
Reverse transcriptase makes DNA from an RNA template
DNA produced is called cDNA (complementary DNA)
How do you make DNA fragments using restriction endonuclease enzymes
Some sections of DNA have palindromic sequences
Restriction endonucleases recognise specific palindromic sequences and cut the DNA at these places
DNA sample is incubated with the specific restriction endonuclease which cuts the DNA fragment out via a hydrolysis reaction
Sometimes the cut leaves sticky ends (unpaired bases at the end of the fragment)
They can be used to bind/ anneal the DNA fragment to another piece of DNA that has complementary sticky ends
How do you make DNA fragments using a gene machine
a database containing the necessary info to produce the DNA fragment is used
The sequence that is required is designed
The first nucleotide in the sequence is fixed to a support
Nucleotides are added step by step in correct order
Protective groups are added (make sure nucleotides join at correct point and prevents unwanted branching)
Short sections of DNA called oglionucleotides (roughly 20 nucleotides long)
How do you make DNA fragments using a gene machine
a database containing the necessary info to produce the DNA fragment is used
The sequence that is required is designed
The first nucleotide in the sequence is fixed to a support
Nucleotides are added step by step in correct order
Protective groups are added (make sure nucleotides join at correct point and prevents unwanted branching)
Short sections of DNA called oglionucleotides (roughly 20 nucleotides long)
They are then broken off from the support and protecting groups are removed
Oglionucleotides can then be joined together to make longer DNA fragments
How do you amplify the DNA fragments you’ve just obtained
DNA fragments inserted into a vector
The vector transfers the DNA fragment into host cells
Identifying transformed host cells
To produce proteins you need a promotor and a terminator region
How is the DNA fragment inserted into a vector and how is recombinant DNA produced
A vector is something that can be used to transfer DNA into a cell
They can be plasmids or bacteriophages
The vector DNA is cut open using the same restriction enzyme endonuclease that was used to isolate the DNA of the target gene
So sticky ends of the vector are complimentary to the sticky ends of the DNA fragment containing the gene
The vector DNA and DNA fragment are mixed together with DNA ligase
DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA
This process called ligation
The new combination of bases in the DNA (vector DNA and DNA fragment) is called recombinant DNA
what is ligation
DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA
How does the vector transfer the DNA fragment into host cells
The vector with the recombaint DNA is used to transfer the gene into cells
If a plasmid vectors used host cells have to be persuaded to take in the plasmid vector and its DNA
With a bacteriophage vector the bacteriophage will infect the host bacterium by injecting its DNA into it
The phage DNA (with the target DNA in it) then intergrates into the bacterial DNA
Host cells that take up the vectors containing the gene of interest are said to be transformed
How do we identify transformed host cells
Marker genes are used to identify the transformed cells
Marker genes can be inserted into the vector at the same time as the gene to be cloned
Host cells are grown on agar plates
Each cell divides and replicates its DNA creating a colony of cloned cells
The marker gene can code for antibiotic resistance
Or it can code for fluorescence where when placed under UV light the transformed cells glow and are then easily identified
what are promotor regions
promotor regions are DNA sequences that tell the enzyme RNA polymerase when to start producing mRNA
what are terminator regions
Terminator regions tell the enzyme when to stop
Without the right terminator region, it won’t stop in the right place and a different protein will be coded for
what does PCR stand for
polymerase chain reaction
Can be used to make millions of copies of a fragment of DNA in just a few hours
what are the 3 stages of PCR and their temperatures
Denaturation 95 degrees
Annealing 55-70 degrees
Extension 72 degrees
what happens in the denaturation stage of PCR
H bonds of DNA are broken, 2 strands of DNA are separate
what happens in the annealing stage of PCR
Primers bind specifically to their complementary sequence in the single stranded DNA
what happens in the extension stage of PCR
DNA polymerase extends the primers forming new DNA. Uses original DNA as a template, complimentary base pairings with use of covalent bonds to join bases.
What are the reagents needed for PCR
DNA sample (template)
Primers- to direct the synthesis of the specific target region
Buffers
DNA polymerase – catalyst
Deoxynucleotide triphosphates- contain the bases complimentary to the DNA
