TOPIC 8.4 gene technology Flashcards
recombinant DNA
DNA fragment of 2 diff organisms combined bc genetic code is universal (DNA accepted by 2 diff species and funct normally) + transcription translation mechanisms are similar
process of making protein using DNA tech (5 steps)
- isolation of DNA fragments that hv the gene for desired protein
- insertion into organism using vector (plasmids)
- transformation - transf DNA into host
- identification of host cells w new DNA fragment
- growth /cloning of host cell pop
STEP 1. method of producing DNA fragments (1): mNRA to cDNA
mNRA isolated -> combined w free DNA nucleotides + reverse transcriptase -> makes DNA from RNA template (complementary to DNA)
DNA polymerase convert cDNA (single stranded) to DNA (contains target gene)
STEP 1. method of producing DNA fragments (2): cut fragment containing desired gene from DNA
restriction endonuclease cuts ds at palindrome seq
between 2 opp bp = blunt ends
uneven cut = strand exposed unpaired bases = sticky ends
if same RE to cut 2 DNA fragments - complementary
STEP 1. method of producing DNA fragments (3): gene machine to create gene
base seq determined from desired a.a seq -> mRNA codons & complementary DNA triplets worked out
seq required is designed -> series of oligonucleotides (small overlapping nucleotide strands)
joined 1 nucleotide at time added -> join = gene (no introns) replicated by PCR
in vivo cloning adv
STEP 2. invivo: prep DNA fragment for INSERTION
RNA polymerase (and TF to begin transcription) attach to promoter region
mRNA synthesised
RNA polymerase released to end transcription = terminator region
STEP 2. invivo: INSERT DNA fragment to vector DNA
vector DNA isolated, same RE cuts plasmid
vector DNA sticky ends complementary to DNA frag sticky ends
DNA ligase joins sticky ends = recombinant DNA
STEP 3. invivo: TRANSFORMING cells
reintroduce recombinant plasmids to bacteria cell (mix plasmids, bacteria cells, Ca2+ at changing temps) - makes bac memb more permeable to plasmids so can enter cytoplasm
BUT plasmid can close, DNA can coil to form mini plasmid
STEP 4. invivo: IDENTIFYING transformed cells (1) antibiotic resistance marker genes
STEP 4. invivo: IDENTIFYING transformed cells (2) fluorescent markers
transf jellyfish gene into plasmid - produces green fluorescent protein
can quickly identify using UV light
STEP 4. invivo: IDENTIFYING transformed cells (3) enzyme markers
STEP 5. invitro: PCR
mixture of DNA frag, free nucleotides, DNA polymerase & primers
heated to 95C = breaks H bonds = separates ds DNA (removes DNA present as this will be replicated)
primer = short seq of nucleotides complementary to bases at start of frag
cooled to 55C so primers bind to DNA at complementary bp
inc temp to 75C - DNA polymerase joins nucleotides to produce complementary strand/s of DNA
2 new copies of org frag made
rev transcriptase produces cDNA from mRNA
adv invitro cloning
DNA probe
short single strand DNA w label (fluroescent or radioactive)
has bases complementary to base seq of target allele/gene/DNA
