DNA analysis + DNA sequencing Flashcards
(ADD) Semester 1 year 1
Describe electrophoresis
-technique used to separate DNA fragments based on size
-largest fragments move least + smallest fragments move further
-dye used to visualise DNA - fluoresces under UV light
What are 2 ways in which DNA can be analysed?
-using restriction enzymes
-using PCR
Describe how you can analyse DNA using restriction enzymes
-digest vector + gene with same restriction enzyme (RE1) + insert gene into vector
-vector contains own restriction enzyme (RE3) as does the gene (RE2)
-possible cloning outcomes: vector + recombinant DNA (correct), empty vector, recombinant DNA incorrectly oriented in vector
How do you distinguish between the possible outcomes of cloning when analysing DNA using restriction enzymes?
-digest the outcomes with RE1 - determines which vector is empty
-digest the remaining 2 outcomes with RE1 + RE2 - gives a different pattern of restriction enzyme digest fragments
Describe how you can analyse DNA using PCR
-clone inserted using same restriction enzyme (RE1) for both gene + vector
-design 2 primers specific to vector + gene
-1 outside where gene will be inserted in vector + 1 in gene
-both act in opposite direction
-possible outcomes: primers face each other after insertion (correct), empty vector with 1 primer, gene incorrectly orientated in vector (primers act in same direction)
What are the outcomes of PCR when using PCR do analyse DNA?
-correct gene insertion into vector = PCR product - pair of primers
-Empty vector = no PCR product - only 1 primer
-incorrectly oriented gene = no PCR product - primers amplify in same direction
What are the uses of PCR?
-modern genetic fingerprinting - amplifies regions containing repeats
-identification of repeat expansions - caused by certain diseases
-identification of genomic rearrangements - due to cancer
What can restriction fragment length polymorphism (RFLP) determine and what does it require?
-determine a mutation within a restriction enzyme site - need PCR + restriction enzyme digest
-requires a way to detect specific DNA fragment e.g southern blot
What does DNA sequencing rely on us being able to work out?
-how long a DNA molecule is (base pairs)
-what the last nucleotide is
How does DNA sequencing work?
-specialised form of DNA synthesis
-DNA is amplified
-for each fragment, we know the last nucleotide + can measure DNA size
What is a dNTP?
Deoxynucleoside triphosphate
What is a modified dNTP?
-dideoxynucleoside triphosphate (ddNTP)
-can’t form a phosphodiester bond with the 5’ phosphate of next nucleotide
-results in termination of sequence
Describe modern automated DNA sequencing
-1 reaction contains all 4 ddNTPs with different fluorescent label
-capillary gel electrophoresis is read by a computer
-taq DNA polymerase is a PCR-like reaction
Why is modern automated DNA sequencing better than the old version?
-more sensitive + specific
-easy, quick + cheap
What are the components of a sequencing reaction?
-DNA polymerase
-dNTPs + ddNTPs (fluorescently labelled)
-buffer (incl. Mg)
-template DNA - plasmid/PCR product
-primer
