A2 - gene tech Flashcards
producing DNA fragments - reverse transcriptase and restriction endonucleases
reverse transcriptase => uses mRNA in the cytoplasm of a specialised cell as a template for the production of complementary DNA
restriction endonucleases => cut specific sections of DNA to produce ‘sticky ends’ these allow this DNA fragment to be inserted into a plasmid for cloning. specific to a recognition site
in vivo gene cloning (bacterium)
when the DNA fragment is produced we put it into a bacterial plasmid known as a vector (to stop it being digested by enzymes in the cell)
place the plasmid back into the bacterial cell, hopefully the bacterial cells divide (binary fission) producing large amounts of your DNA fragment
dna ligase used to join phosphodiester bonds in plasmid/fragment merge
in vitro cloning (PCR)
reaction mixture: DNA sample, primers (short polypeptide chains), free nucleotides, DNA polymerase
- heat to 95°C to break hydrogen bonds and separate strands
- lower temp to 55°C, so complementary primers can anneal (bind) to the strands
- temp to 72°C as this is temp DNA polymerase works at. it joins to the primers, free nucleotides join up, DNA polymerase creates a copy of the sample by complementary base pairing.
in vivo vs in vitro
VIVO:
accurate
produces recombinant DNA
cuts out specific genes
no risk of contamination
VITRO:
rapid
no living cells needed
dna probes
short single stranded DNA molecule that is complementary to a specific base sequence.
DNA labelling of the fragments either uses radioactive isotopes or fluorescent dye which glows under certain wavelengths of light
uses to detect heritable conditions of health risks
how dna probes work
- fragment of DNA with complementary bases to the mutant allele of the gene is produced
- DNA probe is formed by fluorescently labelling the DNA fragment
- PCR techniques used to produce multiple copies of the DNA probe
- probe is added to single-stranded DNA fragments from person being screened
- if donor has mutated gene, some donor DNA fragments will have a base sequence complementary to the probe and the probe will bind to its complementary bases on the donor DNA
- these DNA fragments are now labelled with the probe and can be distinguished
- if complementary fragments are present, DNA probe will be taken up and the dye will fluoresce (detected by a microscope)
genetic fingerprinting - gel electrophoresis
- extract DNA and increase amount using PCR
- restriction enzymes cleave DNA into smaller segments of various sizes
- DNA placed onto gel and voltage applied.
- small fragments move further towards +ve electrode than large fragments
- fragments transferred to nylon membrane
- radioactive DNA probes added to label the fragments.
- placed onto an X-ray film.
-developing this reveals dark bands where radioactive DNA probes have attached
recombinant dna - why can we pass genes between organisms
the genetic code is universal so the same codons code for the same amino acids in all living things
steps to genetically engineer an organism (basic)
- identify DNA fragment or gene
- isolate it
- multiply it (using PCR)
- transfer into the organism using a vector (eg plasmid, virus, liposome)
- identify cells with new DNA fragment (by using a marker) which is then cloned
promoter region
region of DNA that is the binding site for RNA polymerase
the nucleotide bases of promoter region attach both RNA polymerase and transcription factors so begin transcription
(if we want bacteria to transcribe DNA we need to have promoter regions)
terminator region
region of DNA that releases rna polymerase and ends transcription
again must be added to the other end of our dna fragment to stop transcription at the appropriate part
the process of reintroducing plasmids into bacterial cells
called transformation
needs calcium to increase permeability of bacteria
how to tell if bacteria is successfully transgenic (replica plating and antibiotic resistance)
replica plating/antibiotic resistance => bacteria containing plasmids are resistant to ampicillin so survive
bacteria not containing them die
once you have bacteria with plasmids, to see if it’s recombinant or not, do replica plating and add another antibiotic to one plate
bacteria that die contain recombinant DNA, so we know which ones we want on the other plates
fluorescent markers - plasmids
GFP gene inserted into plasmid
human DNA fragment inserted into this GFP gene
this stops it fluorescing, so ones that don’t fluoresce have human genes in them
enzyme marker - plasmids
gene coding for lactase into plasmid
add human DNA fragment to gene coding for lactase (blocking it from hydrolysing lactose)
lactase turns substrate blue
if it goes blue we don’t want it, if it stays colourless it contains human DNA
