19- gene technology Flashcards
recombinant DNA
DNA that is altered to contain nucleotides from two different organisms -> transgenic organism
genetic engineering
deliberate manipulation of genetic material to modify specific characteristics of an organism. involves gene transfer for expression
main ways gene can be obtained for genetic engineering
- made from mRNA of donor using reverse transcriptase
- cut from DNA of donor organism using restriction enzymes
- synth from nucleotides (gene machine)
vector (plasmid/virus/liposome)
carrier used to deliver gene to organism’s cells
restriction enzymes
recognise specific palindromic sequences in DNA
cut DNA at sites through hydrolysis reaction
why do diff REs cut diff DNA sequences
each has active site complementary to specific DNA sequence, so only cuts that unique recognition sequence
how can REs isolate specific DNA fragment
if recognition sequences are at either end of desired DNA fragment, REs cut these to separate fragment from rest of DNA
obtain desired gene
sticky ends
short, overhanging sequences of unpaired base sequences of DNA fragment cut by REs
used to insert genes into vectors
how is cDNA made, and how is it used to generate DNA
mRNA -reverse transcriptase> sscDNA -DNA polymerase> dscDNA
how can genes be synthesised without template DNA
- choose codons for desired amino acid sequence from known protein structure
- use computer to direct synthesis of short DNA fragments(oligonucleotides) in DNA synthesiser machines
- join fragments tgt to make longer sequence of nucleotides, forming desired gene
- PCR constructs cDNA strand+amplifies gene to produce multiple copies
key stages of gene transfer
- desired gene identified+isolated
- multiple copies of gene made using PCR
- gene inserted into vector
- vector delivers genes into cells
- cells with new gene identified, by markers
- cells with new genes are cloned
DNA ligase forms phosphodiester bonds between
sugar + phosphate groups of 2 DNA strands
join sticky ends of isolated DNA fragment to sticky ends of DNA in plasmid vector
why is same RE used to extract DNA fragment + on DNA in vector
ensures sticky ends are complementary so isolated DNA fragment+DNA of vector can be joined
what is commonly used as vector in genetic engineering
plasmid/bacteriophage
how to identify whether bacteria have been successfully genetically modified using marker genes (4)
- marker gene causes them to exhibit specific trait(antibiotic resistance- incorporated into vector)
- marker gene coding for fluorescent protein can be seen under UV light, (GFP incorporated into vector)
- marker gene inserted within GFP gene, preventing fluorescence if successfully incorporated
- marker gene may be incorporated that codes for enzymes that cause colour change to particular substrate
what are promoters in DNA and why are they relevant to genetic engineering
region where RNA polymerase binds to, initiating transcription
promoter regions added to isolated DNA fragments so RNA polymerase can bind+transcribe a gene
gene editing
altering genome by: deleting, inserting, replacing DNA sequences at specific sites
potential applications of tools like CRISPR/Cas9
- disease treatment
- preventing disease transmission
- industrial applications
what is PCR
method to amplify DNA fragments rapidly+efficiently
why does specific type of DNA polymerase need to be used
Taq polymerase- ability to withstand high temperatures without denaturing
role of primers in PCR
add short nucleotide sequences binding to complementary DNA strands, initiating replication
basic components needed for PCR
- DNA fragment as template
- primers
- DNA polymerase
- free nucleotides
- thermocycler
PCR
- denaturation(95°C): break H bonds between complementary strands+separate
- annealing(55°C): cool so primers can bind to DNA strands, forming H bonds
- extension(72°C): DNA polymerase adds free nucleotides to ends of primers, extending DNA strand to form complete copy
+: PCR
rapid speed: not possible in in-vivo cloning
precision: automated nature of thermocycling ensures accurate amplification of DNA fragment
low DNA needs:
no cells needed: simpler
