Molecular biology 3 - recombinant DNA technology Flashcards
What is recombinant DNA?
Joining together of DNA molecules from different organisms (which is inserted into a host)
Applications of recombinant DNA technology
Vaccine production, protein therapies, pharming, gene therapy, transgenic animals
Examples of protein therapies produced using recombinant DNA technology
human insulin, human growth hormone, interferon (cytokines)
Example of pharming
Transgenic sheep producing blood clotting factors in their milk to treat haemophilia
How can gene therapy be used to treat/prevent disease?
By replacing the mutated gene with a healthy one or adding a new (dominant) gene into genome.
What are transgenic animals?
animals that possess an integrated gene or DNA sequence in their genome, which can be passed onto offspring
How can transgenic animals benefit farming?
Improve the reproductive performance, increase growth rate, improve carcass composition, improve milk production, increase disease resistance
What are the 2 methods used to isolate and amplify a gene?
gene cloning (in vivo) and PCR (in vitro)
Outline of gene cloning
- gene is isolated by restriction enzyme
- Gene is separated from other fragments by gel electrophoresis
- Plasmid cut by same restriction enzyme
- Sticky ends of gene and plasmid anneal by complementary base pairing
- annealed ends covalently joined by DNA ligase
- plasmid inserted into bacterium host
- bacteria grows into colony
- cloned cells are lysed
- plasmids isolated by centrifugation
- gene reisolated using restriction enzyme
What are restriction enzymes?
Enzymes that cut double-stranded DNA at specific DNA sequences (recognition sites)
Features of recognition sites (where restriction enzyme cuts)
4-6 bp long and palindromic
Name of staggered cut made by most restriction enzymes
sticky ends (some leave blunt ends)
After the sticky ends anneal, how are the fragments joined by covalent bonding?
Using DNA ligase
5 general procedures required for cloning DNA
- isolating DNA to be cloned
- selecting a cloning vector e.g. plasmid
- covalent joining of DNA fragments by DNA ligase
- moving recombinant DNA into host
- identifying hosts that contain recombinant DNA
What is a vector?
a carrier or delivery agent
What is a cloning vector?
small molecule of DNA capable of autonomous replication
How are host cells containing the recombinant DNA identified?
The plasmid contains 2 antibiotic resistance genes (ampicillin and tetracycline resistant genes). The foreign DNA is inserted into the tetracycline gene so the bacteria with the recombinant plasmid should be resistance to ampicillin but sensitive to tetracycline. The bacteria are grown on plates containing ampicillin to select for bacteria that have taken up the plasmid. The colonies are then transferred into identical position on two plates: one with ampicillin and tetracycline and one with ampicillin. The colonies that grow only on the ampicillin plate contain the recombinant plasmid.
What does the Polymerase Chain Reaction do?
Amplifies specific DNA sequence between two known flanking sequences (used to prepare primers)
What are primers?
short, single stranded DNA molecules complementary to the ends of a defined sequence of DNA template
Uses of PCR
used to identify microbial DNA in clinical samples (using primer), amplifies DNA in forensic samples etc
What is required for PCR?
The double stranded DNA to be amplified (template), primers, dNTP, Taq DNA polymerase
3 stages of a PCR cycle
- Denaturation - separates 2 strands of template DNA (94C)
- Annealing - primers bind to complementary sequences on template DNA (50-60C)
- Extension - primers extended by Taq DNA polymerase (72C)
(repeated 20-40 times)
After n cycles, how many copies of the original template DNA is produced?
2^n (doubles after each cycle as each new strand becomes template in next cycle)
What are the base sequences at the ends of the PCR product?
The primer sequences
Advantages of PCR
- fast
- primers are very specific so only target DNA is replicated
- only needs one template DNA molecule (used in forensics)
Steps of gel electrophoresis
- DNA fragments placed in well in agarose / polyacrylamide gel
- current applied
- DNA (-ve) moves towards anode (+)
- Smaller DNA fragments move further
- Autoradiography / fluorescent dye used to visualise DNA
Example of stain used to visualise DNA in gel electrophoresis
ethidium bromide (illuminated with UV irradiation to become fluorescent)
How is ethidium bromide used to detect DNA?
Ethidium bromide intercalates between planar rings of DNA double helix. Fluoresces under UV irradiation.
What is DNA sequencing?
process used to determine order of bases in DNA (gene or genome)
Alternative name for Sanger sequencing
Dideoxynucleotide chain termination method
What is required for Sanger sequencing?
Single-stranded DNA template, primer, dNTP, ddNTP, DNA polymerase, label (e.g. primer of ddNTP)
Function of DNA polymerase
Catalyses DNA strand synthesis
How do dideoxynucleotides (ddNTP) cause chain termination?
3’C has a H instead of an OH group so a phosphodiester bond cannot form to the next nucleotide.
Why are ddNTP added at a much lower concentration than dNTP?
ratio of 1:10 - 1:300 (depends on number of bases in fragment) allows sufficient strand elongation for analysis
Which end of the DNA does the primer bind to?
3’ end
Procedure of Sanger DNA sequencing
- DNA mixed with primer
- primer binds to 3’ end of DNA
- DNA-primer mixture divided into 4 reaction tube, each containing one of ddATP, ddGTP, ddCTP, ddTTP
- Chain synthesis catalysed by DNA polymerase
- Gel electrophoresis
- DNA bands detected by autoradiography or laser
- DNA sequence deduced
How is the DNA sequence deduced using Sanger sequencing?
Each lane in gel electrophoresis consists of a different ddNTP. Dark bands indicate where chain termination has occurred and therefore the location of the specific base. DNA sequence is read from bottom-up across all 4 lanes. DNA sequence of the original strand is complementary to the one inferred from the gel.
Name of an automated Sanger throughput sequencing instead of gel-based
dye terminator sequencing (DNA fragments with dye terminators pass through the capillary tube and the colour is detected by a laser)
Comparison of gel based vs automated Sanger sequencing throughput
Automated can sequence more base pairs (750-1000bp) and more samples (96) per run than gel-based (250-500bp and up to 8 samples)
Name of technology that can sequence millions of genes at once
high throughput / next generation sequencing (NGS) - requires bioinformatics analysis
Advantage of using high throughput / next generation sequencing
Cheap and rapid
