Recombinant DNA Flashcards
Uses of recombinant DNA technology
Combining different organism’s DNA allows scientists to manipulate + alter DNA to improve industrial process + medical treatment
Why does recombinant DNA work?
The genetic code is universal = transcription/translation occur by same mechanism and = the same aminos across all organisms
3 methods of DNA fragmentation
1) Reverse transcription
2) Restriction endonuclease
3) Gene machine
Describe DNA fragmentation with reverse transcriptase
- Found in HIV and it makes DNA copies from mRNA
- Cell that naturally produces protein of interest is selected
- Cells should have large amount of mRNA for the protein
-mRNA acts as a template and free nucleotides with complementary bases align and are joined by the reverse transcriptase - Single-stranded cDNA is made
- DNA polymerase used = double stranded
Advantage of reverse transcriptase
- cDNA is intron free as it is based of mRNA template
- mRNA of interest available to make cDNA
Disadvantage of reverse transcriptase
Most steps = more time consuming + difficult
Describe DNA fragmentation with restriction endonuclease
- Occur naturally in bacteria as a defense mechanism = enzyme that cut up DNA
- Enzyme has complementary AS to DNA bases =recognition sequences = each enzyme cuts at a specific location
- Some enzymes = cut in same location in double strand = blunt end
- Other enzyme = cut to create staggered ends = exposed DNA bases = palindromic = sticky ends
- Sticky ends join to DNA with complementary base pairing
Advantages of restriction endonuclease
Sticky ends make it easier to insert DNA
Disadvantages of restriction endonuclease
Still contains introns
Describe DNA fragmentation with gene machine
- Created in lab using computerized machine
- Scientist examine protein of interest to identify amino acid sequence = work out mRNA/DNA sequence from it
- Enter DNA sequence into computer to check for biosafety of protein
- Computer creates small sections of overlapping single strands of nucleotides = oligonucleotides
- Oligonucleotides joined = DNA
Advantages of gene machine
- Quick
- Accurate
- Intron free
- Can design exact DNA fragments wanted with sticky ends + labels + preferred codons
Disadvantages of gene machine
Need to know the amino acid sequence
Types of fragment cloning
1) In vivo cloning
2) In vitro cloning
Steps of in vivo cloning
1) Inserting DNA fragment into vector
2) Transform host cells with the vector
3) Identifying transformed cells
4) Growing clones of host
Describe modification of DNA fragment before insertion
- DNA fragment must be modified to ensure transcription
- Promoter region: Sequence of DNA added to start of fragment = binding site for RNA polymerase = transcription
- Termination region: Sequence of DNA added to the end of fragment = RNA polymerase detach = stop transcription = only 1 gene copied into mRNA at a time
Describe insertion of DNA into vector in in vivo cloning
- Same restriction endonuclease used to cut plasmid
- Same sticky ends
- DNA fragment sticky ends are complementary to plasmid sticky ends
- Annealing = both combined using ligase enzyme = catalyzes condensation reactions = phosphodiester bonds between nucleotides
Describe transformation of host cell in in vivo cloning
- Vector to be inserted into host cell
- Cell membrane made more permeable by added Ca2+ ions + heat shock = increased permeability
- Vector enters host cells cytoplasm
Reasons host cell doesn’t take up recombinant plasmid
1) Recombinant plasmid doesn’t enter host cell even with increased permeability
2) Plasmid rejoins before DNA fragment entered
3) DNA fragment sticks to itself rather than plasmid
Use of marker genes
- Used to identify which bacteria successfully take up recombinant plasmid
- 3 types:
1) Antibiotic-resistant genes
2) Genes coding for fluorescent proteins
3) Genes coding for enzymes
Describe identification with antibiotic-resistant genes
- Plasmid contains tetracycline + ampicillin-resistant genes
- DNA fragment inserted = tetracycline-resistant gene disrupted = gene no longer able to create functional protein
- Grow bacterial colonies on agar
- Transfer to plate with ampicillin antibiotics = see which colonies are still left = ampicillin-resistant
- Transfer to plate with tetracycline antibiotics = see which colonies left = tetracycline-resistant = not host containing recombinant DNA
Describe identification with fluorescent markers
- Jellyfish contain green fluorescent protein = GFP
- Inserted into plasmid
- DNA fragment inserted into middle of gene = disrupts = prevents production of GFP
- Check under UV light = non-glowing colonies = contain recombinant DNA
Describe identification with enzyme markers
- Lactase turns substances from colorless to blue
- Enzyme gene inserted into plasmid
- DNA fragment inserted into middle = disrupted = prevents production of enzyme
- Colonies grown on agar with a colorless substance = colonies that can’t turn blue = contain recombinant DNA
Steps of in vitro cloning
1) PCR
Equipment needed for PCR
1) Thermocycler: Allows temperature changes over and over again
2) DNA fragment that needs to be amplified
3) DNA polymerase/taq polymerase: From bacteria in hot springs = high optimum temperature
4) Primers: Complementary short sequence of single strands attached to start + end of fragment
5) DNA nucleotides
Describe the method for PCR
- DENATURING: Temperature increased to 95°C = break H-bonds = split DNA into single strands
- ANNEALING: Temperature dropped to 55°C = allow primers to collide + attach with complementary sequence
- SYNTHESIS: Optimum temperature is 72°C for taq/DNA polymerase which attaches complementary free nucleotides = makes new strand aligned next to each template
Advantages of PCR
1) Automated = more efficient
2) Rapid = 100 billion copies of DNA made in hours
3) Doesn’t require living cells = quicker + less complex techniques needed
What is VNTR?
- 95% of DNA is introns which consist of variable number tandem repeats
- The more closely related you are the more similar your VNTR
What is genetic fingerprinting?
Analysis of VNTR DNA fragments to determine genetic relationships + genetic variability in a population
Steps of gel electrophoresis
1) Extraction
2) Digestion
3) Separation
4) Hybridization
5) Development
6) Analysis
Describe extraction
- Only need a tiny amount e.g drop of blood or hair follicle
- Extract DNA by separating from rest of cell
- Quantity can be increased through PCR
Describe digestion
- Restriction endonuclease is used to cut DNA into smaller fragments
- Use enzyme that is complementary to DNA before + after target VNTR
Describe separation
- DNA samples loaded into wells in agar gel
- Agar gel placed in buffer solution with electrical voltage applied
- DNA phosphate backbone is negative = move through gel towards positive end
- Agar gel = resistance = smaller DNA move through gel faster = different lengths of DNA separated
- Alkaline added to separate double strand
Describe hybridization
- Radioactive/fluorescent probes complementary to base sequence of VNTR added
- Different probes added to singe stranded VNTR = binding
Describe development
- Transfer agar to nylon sheet to prevent drying + cracking
- Expose nylon sheet to X-rays/UV light to visualize positions of probes = where VNTR are
Describe analysis
DNA bands compared to identify genetic relationship/disease-causing gene/DNA from crime scene
