DNA technologies Flashcards
wk 12
What is PCR?
Technique that Amplifies single or few copes of DNA across several orders of magnitude
What are the three reactions for PCR?
DENATURATION: heat to 95 degrees to denature into two separate strands
ANNEALING: cooling to allow primers to bind to template
EXTENSION: Temperature increased to optimum temp for DNA polymerase
What is SYBR Green?
= double stranded DNA-binding dye that intercalates into double-stranded DNA (dsDNA)
What are the two methods of choice for nucleic acid quantification?
PCR and qPCR
What is SYBR Green used for?
A dye that allows for the measurement of the amount of PCR product.
As amplification proceeds = number of SYBR green molecules increase (fluorescence is proportional to amount of products accumulated.
What is a restriction enzyme?
Enzyme that cuts DNA at restriction site
What does PCR require for aplification?
Two primers (single strand DNA with complementary sequences to ends of DNA want to amplify)
Free dNTPs: deoxynucleotides triphosphates (building blocks of new strands)
Buffer: keep enzyme working
Magnesium ions: cofactor for enzyme
DNA polymerase: catalyses synthesis of new DNA strands
What is the old method used to measure PCRs?
PAGE - agarose gel
Describe the use of agarose gel in PCR meaurement.
It is used to visualise the PCR fragments
they pass electrical current through agarose gel
DNA (negative) moves towards positive terminal
Distance it moves is dependent on number of base pairs
More base pairs = heavier and slower movement
Compare movement against a ladder of DNA fragment lengths
Is DNA positive or negative?
negative
What is the new method for DNA amplification?
qPCR
what is a Palindromic sequence of DNA?
= sequence of one strand of DNA is the same as the other read backwards.
How do restriction enzymes and palindromic sequences create new strands of DNA?
Restriction enzyme cleaves DNA into two strands with overhanging sticky ends
e.g. EcoRI 5’GAATTC’3 and 3’-CTTAAG-5” : Pstl cuts between A and G and cretaes two four base 3’ overhangs
How is the plasmid in cloning vectors cut?
with a restriction enzyme
What makes up a Cloning Vector?
= small piece of DNA + plasmid (or cell of higher organism) + foreign DNA
Describe the DNA used in a cloning vector
The DNA must be from another organism (virus) and be stably maintained in an organism.
describe the basic process of a cloning vector?
Plasmid cut with restriction enzyme
DNA template replicated with PCR (with primers of a specific restriction site)
Product is ‘glued’/ligated into an expression vector with matching site
The plasmid makes a protein –> overexpression
What are three examples of the use of cloning vectors?
Pig insulin (old technique)
Recombinant human insulin production (new technique)
Humanised antibodies
Outline the process of recombinant human insulin production.
Human gene isolated
Bacterial plasmid isolated and cut with restriction enzyme
Human insulin gene inserted using DNA ligase
Vector used to create recombinant bacterium
Bacterium grows in fermentation tank
Insulin extracted and purified
What are the two humanised antibodies used as recombinant protein and therapy
Herceptin and Avastin
What is Macular degeneration?
Degeneration of the retina (subretinal haemorrhage)
Can be seen in diabetes mellitus (high BGL = vascular endothelial growth factors cause blood vessel growth = lifted up retina, leak of blood and fluid and central vision loss.
Discuss the role of Avastin on Macular Degeneration.
Slows progression of disease considerably
Avastin prevents VEGF from binding to the membrane and causing angiogenesis (growth of blood vessels)
What is EGFR and what does its over-expression result in?
Transmembrane tyrosine kinase glycoprotein activated by EGF
Overexpression = tumours of breast, lung, colon, cervix, ovary, oesophagus and endometrium.
what is the relationship between herceptin and cancer?
Provides a target for the immune system to destroy cancer cells that it is attached to
Lower metastases and cancer growth when used
What us Herceptin?
Immune targeted therapy
What does Herceptin and Avastin treatment prevent and what is the impact of this?
Herceptin and Avastin co-treatment prevents micro vessel and epithelium growth.
There is a worse prognosis for those with higher HER2/VEGF density
What is an example of a virus mediated anti-cancer treatment and how (generally) does it work
T-VEC (engineered virus) provokes immune response to treat advanced melanoma
T-VEC = from herpes simplex virus-1, virus becomes attenuated so no longer causes herpes
What is the old method of DNA sequencing?
Sanger method
What does the Sanger method use?
A single stranded DNA template
DNA primer
DNA polymerase
Normal deoxynucleotides (dNTPs)
Modified nucelotides (dideoxyNTPs: ddNTPs) that terminate DNA strand elongation.
Outline the Sanger Method
Four PCR mixes set up, containing stocks of normal nucleotides plus one dideoxynucleotide (ddA, ddT, ddC or ddG)
As a typical PCR will generate over 1 billion DNA molecules, each PCR mix should generate all the possible terminating fragments for that particular base
When the fragments are separated using gel electrophoresis, the base sequence can be determined by ordering fragments according to length
If a distinct radioactive or fluorescently labelled primer is included in each mix, the fragments can be detected by automated sequencing machines
What are the three types of Next Generation Sequencing?
- Illumina (Solexa) sequencing
- Ion torrent: Proton / PGM sequencing
- SOLiD sequencing
What are the benefits of NGS?
Much more quicker cheaper
revolutionised study of genomics and molecular biology
allowed Genome Wide Association Studies
-detect rare disease
-personalise medicine
-better techniques for cancer treatment
What are Genome Wide Association Studies and what do they allow for?
Rapid scanning makers across set of DNA = find genetic variations associated with a particular disease
Used to develop strategies to detect, treat and prevent disease
What are SNPs?
Positions in genome where some individuals have one nucleotide, and others a different one
Swapped nucleotide may be a marker or elevate risk of disease
SNPs are found in: coding and non-coding regions
Very high frequency: 1 in 1000 to 1 in 300 SNPs
can act as a marker for a gene
