gene technology Flashcards
what are sticky ends?
single stranded sections of DNA that overhang at the end of a double stranded molecule
what does reverse transcriptase do to obtain DNA fragments?
Reverse transcribes mRNA into cDNA
DNA polymerase converts cDNA into a double stranded fragment
why is reverse transcriptase used?
mRNA is in higher abundance so easier to extract
what is a key characteristic of cDNA?
it has no introns as they are already removed in mRNA
what is a gene machine and how does it work
to synthesise genes artificially
by determining the amino acid sequence of the desired protein then identifying mRNA codons and complementary DNA triplets -> single stranded oligonucleotides are joined to form DNA -> PCR makes it double stranded and amplifies quantity
what are the advantages of the gene machine technique?
intron free, quick, accurate
what are 3 ways of obtaining DNA fragments?
restriction endonucleases, gene machine, reverse transcriptase
what do restriction endonucleases do to obtain DNA fragments
cut DNA at specific recognition sites by hydrolysing phosphodiester bonds
recognition sites are palindromic
either produce blunt ends or sticky end
what does PCR stand for?
polymerase chain reaction
what does the mixture used in PCR contain?
DNA fragments
free DNA nucleotides
primers
thermostable DNA polymerase
what are primers?
short sequences of single stranded nucleotides, with a specific base sequence complementary to the fragment being copied → allow DNA polymerase to bind and stimulate DNA synthesis
whats are the steps of PCR?
- separation of DNA stands -> at 95C so hydrogen bonds break
- annealing of primer -> at 55C, primers attach to complementary base sequence
- DNA synthesis -> at 72 (optimum temp for DNA polymerase) -> DNA polymerase attaches → phosphodiester bonds form between free nucleotides which align along the template strand by complementary base pairing
what is gel electrophoresis?
a technique used to separate DNA based on length and charge
what are the steps of gel electrophoresis?
- DNA samples amplified and fragments of obtained using restriction endonucleases
- electric current passed through gel -> DNA attracted to positive electrode -> shorter fragments move further
- DNA bands visualised by fluorescent dye / radioactive label
what does gel electrophoresis rely on?
the negative charge of DNA due to phosphate group -> migrates towards positive electrode in electric field
what are DNA probes?
single-stranded, short sequences of DNA nucleotides with complementary base sequence to the allele being screened for. They are radioactively or fluorescently labelled in order to detect if they bind the the target gene
what are the steps of genetic screening?
1.PCR amplifies DNA; fragments obtained using restriction endonucleases
- fragments separated by gel electrophoresis
- fragments made single stranded using alkali
- DNA probe added -> if allele present, probe will anneal and will be visible when visualised
what is DNA profiling?
a technique used to analyse and compare different DNA samples
what are VNTRs?
variable number tandem repeats -> unique to each individual
repetitive sequences of DNA of the non coding DNA
why is genetic screening used?
diagnose genetic disease ie cystic fibrosis
detect cancer + determine most effective drug
identify health risks and inheritance to encourage lifestyle changes
what are the steps of DNA profiling?
- extraction of DNA (blood, semen sample, hair root) -> amplify with PCR
- specific restriction endonuclease used to cut recognition site close to VNTR sequence
- gel electrophoresis separates DNA fragments containing VNTR sequences
- DNA probes with specific base sequence bind with specific VNTR
- visualisation using xray film / uv light producing a series of bands that can be compared
why is DNA profiling used?
forensic science, genetic relationships (paternity testing), medical diagnosis (personalised medicine)
what does recombinant gene technology involve?
transfer of fragments of DNA from one organism to another
how is the DNA fragment prepared?
restriction endonucleases cut DNA leaving sticky ends
promoter and terminator sequences are added to fragment to enable transcription to be stimulated and terminated by RNA polymerase
