Recombinant DNA technologies Flashcards
3 ways of isolation
Reverse transcription
Restriction endonucleases
Gene machine
3 ways of isolation
Reverse transcription
Restriction endonucleases
Gene machine
Reverse transcription
Cell producing certain protein is selected
Cells have lots of mRNA for the protein
Reverse transcriptase binds complementary nucleotides to mRNA
Forms cDNA
DNA polymerase to make 2x strands
cDNA is intron free
Restriction endonucleases
Enzymes that cut DNA
Enzyme has AS complementary to base sequences
Blunt + staggered cuts
Staggered cuts have sticky ends
Sticky ends are palindromic - same forwards as they are backwards
Gene machine
Identify amino acid sequence and DNA sequence from proteins
Sequence entered into computer - checks safety and ethics
Computer creates small sections of overlapping single stranded nucleotides
-oligonucleotides
Can be joined to create DNA for entire gene
PCR used to amplify quantity
Quick, accurate and intron free
5 steps of in-vivo cloning
Create fragments Insertion Transformation Identify transformed cells Grow host cell
Insertion into vector
Cut plasmid with restriction endonuclease
Creates complementary sticky ends
Ligase binds DNA to plasmids
Transformation of host cell
Vector inserted into host cell CSM made more permeable to plasmids: -mixed with Ca2+ -heat shocked Allows vector to enter host cytoplasm
Why do we need identification
Plasmid doesn’t enter the cell
Plasmid re joins before DNA enters
DNA fragment sticks to itself and not plasmid
Antibiotic resistant markers
DNA inserted into vector into antibiotic resistant gene tetracycline, plasmid also contains ampicillin
Grow on agar
Stamp velvet on agar
Stamp onto new agar plate, genes not resistant to ampicillin die
Repeat on new agar
Genes not resistant to tetracycline die
Plasmids that survived ampicillin but died to tertracycline contain the DNA fragment
Fluorescent markers
GFP in jelly fish
Fragment inserted
Non glowing cells contain fragment
Enzyme markers
DNA inserted into lactase gene
Bacteria grown on agar plate with colourless substrate
Lactase turns colourless substrate blue
In vitro
In non living thing
What is PCR
Polymerase chain reaction
Used to amplify fragments from invitro
PCR equipment list
Thermocycler DNA fragments Taq polymerase Primers DNA nucleotides
Thermocycler process
Temp increase to 95 - breaks H bonds, split DNA into single strands
Temp decrease to 55 - primers can anneal to single strands
Temp increase to 72 - DNA polymerase attaches free nucleotides to single strands, optimum temperature for taq polymerase
Advantages of PCR
Automated - more efficient
Rapid - 100 billion copies of DNA within hours
Doesn’t require living cells - quicker and less complex techniques needed
DNA probes
Sample of patient DNA mixed with marked probes
If patient has allele, probe binds
Can be viewed by x-ray or UV light
Fluorescently or radioactively marked
DNA hybridisation
DNA heated to separate strands
Mixed with complementary sequences
Used to identify certain alleles
Personalised medicine and genetic counselling
Screening allows for medicine based on genotypes
VNTRs
95% of DNA is introns
Low chance of people sharing same VNTRs
Genetic fingerprinting checks genetic relationships and variability in a population
GFP - collection and extraction
Sample of DNA collected, amplified by PCR
GFP - digestion
Restriction endonucleases added to cut close to VNTRs
Enzymes that cut close to target VNTRs are added
GFP - separation
DNA samples loaded in small wells in agar gel
Gel placed in buffered liquid with voltage
DNA is negatively charged so moves toward positive end of gel
-electrogelphoresis, smaller pieces move further
-VNTRs separated
-alkaline used to separate double strands
GFP - hybridisation
DNA probe process
GFP - development
Agar shrinks and cracks, so moved to nylon sheet
Exposed to X-rays for radioactive probes, UV light for fluorescent probes
GFP - analysis
Position of DNA bands are compared to identify genetic relations
