3 Flashcards
What is recombinant DNA technology
Joining together of DNA molecules from different organisms and inserting it into a host to produce new genetic combinations of value to science, medicine, agriculture and industry
What does recombinant DNA tech involve
DNA from different organisms is “cut and pasted” together, producing recombinant DNA
When was the first recombinant DNA molecule produced
1972 by Paul Berg who won a Nobel prize for chemistry in 1980
Why is recombinant DNA tech important
Vaccine production
Protein therapies
-insulin
-growth hormones
-interferon
Production of blood clotting factors to treat haemophilia
To produce these proteins the gene is cloned into a plasmid which is then introduced into a bacterial cell; bacteria produce the protein, which is then purified and used in patients
What is gene therapy
-Replace faulty (mutated) gene with a healthy one and add a new gene into the genome
-Used to treat or prevent disease e.g. cancer diabetes, heart disease, cystic fibrosis, haemophilia
How can DNA tech be used with transgenic animals
-They possess an integrated gene or DNA sequence (transgene) in the genome, which can be passed onto offspring
-Improved reproductive performance, increased growth rate, improved carcass composition, improved milk production and/or quality, increased disease resistance
What was the first mammal to be cloned
Dolly the sheep
What is gene cloning
> It produces large numbers of copies of a particular piece of DNA
Genes are usually cloned by isolating them using restriction enzymes, followed by gel electrophoresis and inserting them into a plasmid
The plasmid is then introduced into a bacterium, and the bacterium allowed to grow to produce large numbers of cells and hence many copies of the gene
The gene can then be re-isolated using the same restriction enzyme
What do restriction enzymes do
Cut double-stranded DNA at specific DNA sequences
EX
Blunt - Hae III
Sticky - EcoR V MORE EFFICIENT
What is Gel electrophoresis
-Used to separate DNA fragments on the basis of their size
-Samples are applied to a gel immersed in buffer and a current is applied
-Negatively-charged DNA migrates from the negative electrode (top of gel) to the positive electrode (bottom of gel)
-Larger DNA fragments migrate more slowly than smaller DNA fragments allowing separation by size
Process of gene cloning
- To insert a gene into a plasmid, a restriction enzyme is chosen that cuts on either side of the gene but not the middle
- The gene is separated from other DNA fragments by gel electrophoresis
- A suitable plasmid is cut at one point using the same restriction enzyme
- The cut plasmid and gene are mixed, and the sticky ends of the plasmid and gene are allowed to anneal by base pairing
- The annealed ends are covalently joined using DNA ligase
- The plasmid, now containing the gene of interest, is introduced into the host bacterium
- The bacteria are grown into a colony, using antibiotic resistance genes in the plasmid to select colonies containing plasmids
- Cloned cells are lysed and the plasmids isolated by centrifugation
- Plasmids are cut with the restriction enzyme, releasing the cloned gene for further studying
Why do we use DNA sequencing
To determine base sequences of DNA
Works out the structure of a gene or an entire genome e.g. human genome project
What is sanger sequencing also known as
Dideoxynucleoside chain termination method (used for small scale projects)
Synthesise new DNA strands which are complementary to a single stranded template strand in vitro (primer extension)
500 base pair stretch - generates 500 different DNA molecules in vitro
What are the components in the manual approach to sanger sequencing
- Single stranded DNA template - unknown sequence used as a template for the synthesis of complementary strand
- Primer -short oligonucleotide serves as a primer from synthesis of comp DNA strand
- dNTPs (deoxynucleotides): dATP dCTP dGTP dTTP (building blocks of DNA)
- ddNTPs (dideoxynucleotides): ddATP ddCTP ddGTP ddTTP (modified nucleotides that terminate DNA strand elongation
- DNA polymerase - enzyme that catalyses DNA strand synthesis
- Label - Fluorescent (IRD800) or radioactive (35S). Required to visualise products. Label primer at 5 prime end
How does chain termination occur
> Interruption of DNA strand synthesis depends on presence of ddNTPs
The 3’ - OH group in dNTPs is replaced by -H in ddNTPs
ddNTPs are incorporated into growing DNA chain but as they lack the 3’ - OH required to form a phosphodiester bond with the nest nucleotide - Chain termination occurs
Synthesis is interrupted at every possible site in a given population of molecules, resulting in hundreds of DNA fragments of varying length
ddNTPs are added at a much lower concentration than the standard dNTPs to allow strand elongation sufficient for sequence analysis
Laboratory procedure of DNA sequencing using sanger method
- DNA to be sequences is mixed with primer
- Primer binds to 3’ end of DNA
- DNA-primer mixture is divided into four separate reaction tubes containing:
-all four dNTPs
-one of the four ddNTPs
-DNA polymerase - Chain synthesis proceeds in each of the four reaction mixtures
- Gel electrophoresis (highly resolving) separation of reaction products - bands corresponding to each position of chain termination appears
- DNA bands detected by autoradiography or by laser in an automated sequencer
- DNA sequence can be deduced from the pattern of bands in the four lanes:
-a dark band in a lane indicates a DNA fragment that is the results of chain termination after incorporation of a ddNTP
-the terminal nucleotide base can be identified according to which ddNTP was added in the reaction producing that band
-the relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence
What enzyme catalysis chain synthesis
DNA polymerase
What is the name for the automated approach of sequencing
Dye terminator sequencing
-each terminal end is coloured corresponding to the base and produce peaks example:
A- blue
C- blue
G- yellow
T- red
Differences between Gel-based and automated sanger sequencing
GEL BASED
-250 to 500 base pairs of sequence per sample
-Up to 8 samples sequenced per run
AUTOMATED (more efficient)
-750 to 1000 base pairs of sequence per sample
-Up to 96 samples sequenced for run
Alternative name for high throughput sequencing
Next Generation Sequencing
Advantages of high throughput (NGS) sequencing
-No need for cloning, highly scalable
-Sequence millions of genes and entire genomes at once
-Cheap and rapid
-Requires substantial bioinformatics analysis
What are the four main DNA sequencing methods used by NGS systems
Pyrosequencing
Sequencing by synthesis
Sequencing by ligation
Ion semiconductor sequencing
Why has the cost of human genome sequencing decreased
Due to the development of sequencing from sanger (manual) to NGS
