Gene Tech Flashcards
Define genome
Entire set of dna
What form does the dna need to be in to sequence the genome
Split into dna fragments
Then put in the right order
Define proteome
All the proteins an org can make
Why is the proteome of prokaryotes easy to find
Don’t have much non coding dna
Why is prokaryote proteome sequencing useful
Identifying antigens on virusues or bacteria to make medication and vaccines
Why is it harder to sequence the proteome of eukaryotes
Lots of non coding dna
And regulatory genes
What are regulatory genes
Genes that tell other genes to be switched on or off
What is recombinant dna technology
Transferring a fragment of dna from org to another
Why can dna from org be transferred to another org in a different species
Dna is universal
So it codes for the same amino acids
Protein synthesis mechanism are similar as well
What are orgs that have had transferred dna inserted into them
Transgenic organisms
Why is mRNA of a gene easier to get than the actual dna
Most cells only have 2 copies of the gene in dna
But lots of copies of the gene in mRNA form
What is reverse transcriptase used for
mRNA -> cDNA (complementary dna)
What is the method for productible dna fragments using reverse transcriptase
mRNA isolated from cells
Mixed with free dna nucleotides
Mixed with reverse transcriptase
mRNA acts as a template to make cDNA
What does restriction endonuclease recognise
Palindromic sequences/recognition sequences
What are palindromic sequences
Antiparallel pairs that read the same in opp directions
What is function of restriction endonuclease
Cut dna
Why does specific restriction endonuclease cut specific sections of dna
The shape of the recognition sequence is complimentary to the as
How do u get a section of dna between 2 palindromic sequences
Incubate restriction endonuclease to cut the palindromic sequences
Leaving the dna fragment
What type of reaction happens when dna is cut by restriction endonuclease
Hydrolysis
What is left by restriction endonuclease
Sticky ends
What are sticky ends
Tails on unpaired dna bases at the end of the fragment
What can sticky ends be used for
Anneal to a fragment with the complementary sticky end
What is the gene machine
Make dna from scratch without a preexisting template
How does the gene machine work
Sequence designed
First nucleotide of the sequence is attracted to a support (bead)
Nuceloutudes added in the correct order with protecting groups
Forming oligonucleotides
Broken off from support
Oligonucleotides are joined together to make a longer dna fragment
What is the purpose of protecting groups
Stop unwanted branching
Make sure the nucleotides join at the right points
What does in Vivo mean
inside a living org
What does amplifying dna mean
Make lots of copies
Describe the method of amplifying dna in Vivo
- Dna fragment inserted into a vector
- Vector dna cut open using the same restriction endonuclease that was used to isolate the dna
- Forms complementary sticky ends to the dna fragment
- Vector dna and fragments are mixed with dna ligase
- Vector dna and dna fragment join together = recombinant dna
- Host cells take up the gene (become transformed)
- Marker genes are inserted into the vector at the same time the dna fragment is added
- Host cells grow on an agar plate. Any colony formed from a transformed cell will all have the recombinant dna and marker gene
- Identify transformed cells eg. Uv light
What is ligase used for
Joins sticky ends of dna fragment to sticky ends of vector dna
=ligation
How do host cell cells take up recombinant dna from a plasmid vector
Host cells placed in ice cold CaCl2 = cell walls more permeable
Plasmids added
Mixture is heat shocked to get the plasmids to be taken up
How is recombinant dna taken up by host cells if the vector is a bacteriophage
The bacteriophage will inject its dna into the host cell
How do u produce proteins from the dna fragment
Vector needs to have specific promotors and terminator regions
What are promoter regions
Tell rna polymerase when to start productions mRNA