8.4 - Recombinant DNA technologies Flashcards
Define recombinant DNA technology
the transfer of fragments of DNA from one organism, or species, to another
Which feature of the genetic code allows recombinant gene technology to be successful (transferred DNA can be translated within cells of transgenic organism)?
universal code, transcription/translation mechanisms similar
Describe the general steps of transferring a gene
isolation: DNA removed from cells
restriction: cut DNA to remove gene
ligation: DNA inserted into vector
transformation: vector put into host cell
selection: isolate cell that contains new gene
replication: copy transgenic organism
Describe how reverse transcriptase can be used to produce DNA fragments
- mRNA isolated from cells + mixed with free DNA nucleotides and reverse transcriptase
- mRNA converted to cDNA (comp. DNA) by reverse transcriptase as it joins DNA nucleotides w/ comp. base pairs to mRNA sequence
- DNA polymerase adds free nucleotides to make gene double-stranded
Describe how restriction endonuclease cuts DNA fragments
- cuts DNA double strand at recognition sequence
- most useful = staggered cuts as they leave sticky ends on DNA, causing ends of 2 fragments to be comp.
- attach, then stronger covalent bonds form
NB: DNA produced contains introns
Define recognition sequence
particular base sequence restriction endonuclease binds to due to active site shape
Describe how a ‘gene machine’ can be used to create genes
- sequence of nucleotide bases designed and fed into computer
- oligonucleotides designed by computer
- assembled by adding 1 nucleotide at a time, and joined to make a gene
- no introns
- replicated using PCR to give numerous copies
- inserted into plasmid
Define oligonucleotide
small, overlapping single strands of nucleotides
Define palindromic sequence
when DNA base sequence is the same forwards and backwards
Describe the polymerase chain reaction (PCR)
purpose: amplifies DNA by making millions of copies of a DNA sample
- reaction mixture: DNA sample, primers, free nucleotides + Taq polymerase
- heated to 92 degrees to break H bonds, separating the 2 strands
- cooled to 55 degrees so primers can bind to strands
- temp. increased to 70 degrees
- Taq polymerase copies sample by comp. base pairing using free nucleotides
- cycle repeated approx. 30x and gives rise to sufficient DNA to create DNA profile
Name the 2 techniques by which DNA fragments can be amplified and the meanings of each
in vivo: in cells inside the organism
in vitro: in cells outside of the organism
Describe in vivo cloning
- same restriction enzyme cuts plasmid and gene (from bacterium) to create comp. sticky ends, then joined
- fragments incubated with plasmids
- base pairing between comp. ends
- then annealed using DNA ligase, forming phosphodiester bonds
- recombinant DNA molecule created
Describe the differences (including advantages and disadvantages) between in-vitro and in-vivo cloning
in-vitro:
- uses PCR
- fast, automated, reliable
- does not require living cells
- problems: contamination, errors
in vivo:
- uses recombinant plasmids
- accurate and useful as gene placed in cells to be expresssed
- problems: time consuming, requires monitoring of cell growth
Define transgenic organism
organism that contains transferred DNA
How are transgenic microorganisms formed?
- electroporation stimulates bacterial cells to take up plasmids
- increases permeability of bacterial membranes
- add Ca2+ and rapid temp. increase
Describe the function of gene markers
- check whether DNA taken up by bacteria
- genes incorporated into plasmid
Examples of gene markers
- antibiotic resistance
- fluorescent protein
- enzyme whose action can be identified (colour change)
Features of replica colonies
- genetically identical
- in exact same position
(as original colony)
Define promotor region
specific sequence of DNA bases transcription factor binds to, initiating transcription
Define terminator region
specific sequence of DNA bases that causes RNA polymerase to detach and stop transcription
Define vector
organism used to carry isolated DNA fragment into host cell
Describe the differences between a DNA probe and DNA primer
- primers used primarily in PCR
- probes are primers with an addition on 1 group that allows us to see the primer
- probes can fluoresce when attached to DNA
Describe how a DNA probe works
- DNA sample digested using restriction endonucleases + separated using electrophoresis
- DNA fragment immobilised and incubated with the labelled DNA probe
- if target allele is present it will hybridise
- membrane is exposed to UV light/X ray - if gene is present there will be a florescent band
Define microarray
screening for many genes at once
