gene technology Flashcards
(33 cards)
what are the 3 ways to obtain DNA fragments
restriction endonucleases
reverse transcriptase
gene machine
how do restriction endonucleases work
cut DNA at specific recognition site to produce either blunt or sticky ends
what are sticky ends
single stranded sections of DNA that overhang at the end of a double stranded molecule
how does reverse transcriptase work and why is it used
create cDNA from mRNA
DNA polymerase converts DNA into a double strand
mRNA is in high abundance and introns are already removed
how does the gene machine work and what are its advantages
artificially synthesises DNA genes -> amino acid sequence of the desired protein determined then complementary mRNA codons and DNA triplets identified
PCR used to amplify quantity
fast, accurate and intron free
what is PCR
polymerase chain reaction
what is in the PCR mixture
DNA fragments
free DNA nucleotides
thermostable DNA polymerase
primers
what are primers and what do they do
short single strands of DNA complementary to the DNA fragment being copied
allow DNA polymerase to bind stimulating DNA synthesis
what happens at 95C in the PCR process
DNA strands are separated -> hydrogen bonds break
what happens at 55C in the PCR process
annealing of the primer -> attachment to complementary base sequence
what happens at 72C in PCR process
optimum temp for DNA polymerase to form phosphodiester bonds between nucleotides
what is gel electrophoresis
a technique used to separate DNA based on negative charge and length
what are the steps of gel electrophoresis
DNA samples amplified and fragmented using restriction endonucleases → mixed with a fluorscent dye / radioactively labelled (to track movement) and loaded into the wells
electric current passed through gel → DNA attracted to positive electrode → shorter fragments move further than longer fragments
DNA bands can be visualised by stain / radioactive label
what are DNA ladders used for
size comparison
what are DNA probes
single-stranded, short sequences of DNA nucleotides with complementary base sequence to the allele being screened for
radioactively or fluorescently labelled to detect if they bind target gene
what does genetic screening used for
diagnose genetic disease
detect cancer, and determine the most effective drug
identify health risks and inheritance → encourage lifestyle changes → however can cause discrimination
what is DNA profiling
a technique used to analyse and compare different DNA samples
what are VNTRs
variable number tandem repeats in non coding DNA that are unique to each individual
what are the steps of DNA profiling
- DNA is extracted (blood droplet, hair root, semen sample) → PCR used to amplify
- specific restriction endonuclease used to cut recognition site close to VNTR sequence
- gel electrophoresis separates DNA fragments containing VNTR sequences → double strands immersed in alkali to separate strands
- DNA probes with specific base sequence bind with specific VNTR → between individuals, fragments will differ in length as different sequence
- visualisation using x-ray film / uv light results in series of bands which can be compared with other samples
what is DNA profiling used for
forensic science
genetic relationships (paternity testing)
medical diagnosis (personalised medicine)
what is a transgenic organism
one whose genome has been altered by introducing a foreign gene due to the universal nature of code
what does recombinant gene technology involve
the transfer of fragments of DNA from one organism to another
how is a DNA fragment prepared before it is inserted into a vector
restriction endonucleases cut DNA, leaving sticky end
promoter and terminator sequences are added to fragment so transcription can be stimulated and terminated by RNA polymerase
what are plasmids
circular DNA molecules used to transport DNA fragment into host cell
