CB14 & CB15 Molecular Tools & Diagnostics Flashcards
What is a restriction enzyme
An enzyme which cuts DNA into fragments at particular sequences
Create “blunt” or “sticky’’ ends
What is DNA polymerase
Enzymes that synthesise the nucleotide chain
5’-3’
What is lipase
An enzyme that catalyses the joining of two molecules by forming a new chemical bond
What is reverse transcriptase
An enzyme that uses mRNA as a template to synthesise new DNA strand
Outline the process of cloning
- Genome & multiple cloning site of vector are cleaved using same restriction endonucleases - same sticky ends
- Resolve via agarose gel & purify fragments
- DNA ligase reaction forms recombinant vector
- Transform bacteria with plasmid
- Select for plasmid using agar containing antibiotic (resistant gene)
- Purify plasmid - to express gene/protein of interest
Explain the process of hybridisation
The process in which two complementary single-stranded DNA/RNA molecules bond together to form a double stranded molecule. The bonding is dependent on the appropriate base-pairing across the two single stranded molecules.
Explain the purpose of PCR
• Selectively & faithfully amplify any DNA template sequence
What is the difference between PCR & qRT-PCR
Endpoint PCR measures amplification at the end of 30-40 cycles
qRT-PCR measures the amount of product amplification after each cycle
Outline the process of DNA sequencing (Sanger sequencing)
• Unknown DNA sequence used as template for PCR
• add DNA polymerase, primers, dNTPs & one type of ddNTP (A,C,G,T)
• DNA polymerase will replicate using nucleotides, but terminates when it reaches ddNTP complementary base pair
• results in many different fragment lengths of DNA
• end of fragments are fluorescently labelled, & separated by high resolution capillary electrophoresis
• creates an electropherogram
Explain some of the useful features of plasmid vectors
“Tag” - allows purification, detection, visualisation of protein of interest
Selectable marker - control expression (off or on)
Antibiotic resistance gene - select only cells containing the vector
Outline the process of protein resolution by gel electrophoresis
• Protein sample must be denatured & reduced to separate out the individual proteins
• add strong detergent, SDS, & heat the mix to denature the proteins, add reducing agent to break disulphide linkages
• separates proteins based on size only
Summarise the process of protein detection using antibodies
Immunoblotting
- after SDS-PAGE separation, electro transfer proteins to a nitrocellulose membrane
- use antibodies to identify individual proteins
- primary antibody binds to protein of interest, secondary antibody used for detection (labeled)
ELISA
- enzyme linked immunosorbent assay
- soluble proteins
- capture target protein in a sample using specific antibody
- target molecules detected & quantified using a labeled enzyme
Explain how a lateral flow test works
- Antibody based technology for rapid, point of care testing
- looking for presence of biomarker (protein) in a sample
- specific capture antibodies bind to biomarker of interest
- detection of the captured antibody/biomarker complex is achieved via additional antibodies in fixed positions on the assay strip
- capture antibodies are enzyme-linked; they catalyse a reaction that produces a dye (visual)
Give examples of how molecular techniques are used in human & veterinary biology
• Looking for biomarkers of health & disease
• parentage testing
• legal cases
• food standards
Explain now PCR can be used in diagnostic testing
- PCR amplification of known DNA sequence within a sample
- amplification of certain genes in individual’s genome
