recombinant dna tech Flashcards
what is recombination dna tech
transfer dna fragments from one organism to another
why can transferred dna be translated within cells of reciptient organism
genetic code is universal
transcription and translation mechanisms are universal
dna fragment production via restriction enzymes
restriction enzymes cut dna at specific bases - recognition sequences
at either side of desired genes
shape of recognition sequence comp to active site
many cut in staggered fashion forming sticky ends
how can dna fragments be produced from mrna
Isolate mRNA from a cell that readily synthesises the protein coded for by the desired gene
2. Mix mRNA with DNA nucleotides and reverse transcriptase
reverse transcriptase uses mRNA as a template to synthesise a single strand of complementary DNA (cDNA)
3. DNA polymerase can form a second strand of DNA using cDNA as a template
adv of obtaining genes from mrna rather than dna
Much more mRNA in cells making the protein than DNA → easily extracted
in mRNA, introns have been removed by splicing whereas DNA contains introns
So can be transcribed & translated by prokaryotes who can’t remove introns by splicing
dna fragments via gene machine
● Synthesises fragments of DNA
quickly & accurately from scratch without need for a DNA template
○ Amino acid sequence of protein determined, allowing base sequence to be established
● These do not contain introns so can be transcribed & translated by prokaryotes
amplify dna fragment via pcr
heat mix to 95
separates dna strands breaking h bonds between bases
mix cooled to 55
allows primers to bind to dna fragment template strand by forming h bonds with Comp bases
mix heat to 72
Nucleotides align next to complementary
exposed bases
DNA polymerase joins adjacent DNA
nucleotides, forming phosphodiester bonds
primers role In pcr
Primers are short, single stranded DNA fragments
Complementary to DNA base sequence at edges of region to be copied / start of desired gene
Allowing DNA polymerase to bind to start synthesis
● Two different primers are required (as base sequences at ends are different)
summarise steps involved in amplifying dna fragments in vivo
Add promoter and terminator regions to DNA fragments
Insert DNA fragments & marker genes into vectors using restriction enzymes and ligases
Transform host cells by inserting these vectors
Detect genetically modified (GM) / transformed cells / organisms by identifying
those containing the marker gene (eg. that codes for a fluorescent protein)
Culture these transformed host cells, allowing them to divide and form clones
why’s promoter region added
Allow transcription to start by allowing RNA polymerase to bind to DNA
Can be selected to ensure gene expression happens only in specific cell types
Eg. in gland cells of a mammal so the protein can be easily harvested
terminator region purpose
ensure transcription stops at end of gene by stopping rna polymerase
role of enzymes inserting dna fragment into vectors
- Restriction endonucleases cut vector DNA
○ Same enzyme used that cut the gene out so vector DNA & fragments have sticky ends that can join by complementary base pairing - DNA ligase joins DNA fragment to vector DNA
○ Forming phosphodiester bonds between adjacent nucleotides
how’s host cell transformed using vector
Plasmids enter cells
● Viruses inject their DNA into cells which is then integrated into host DNA
why’s marker gene added to vector
To allow detection of genetically modified
○ If marker gene codes for antibiotic resistance, cells that survive antibiotic exposure = transformed
○ If marker gene codes for fluorescent proteins, cells that fluoresce under UV light = transformed
As not all cells will take up the vector and be transformed
recombinant dna tech use in medicine
GM bacteria produce human proteins (eg. insulin for type 1 diabetes) → more ethically acceptable than using animal proteins and less likely to cause allergic reactions
● GM animals / plants produce pharmaceuticals (‘pharming’) → cheaper
● Gene therapy
