Sequencing Flashcards
What is DNA sequencing?
Analysis of DNA using molecular biology techniques
What can go wrong with molecular cloning?
- empty vector
- incorrect recombinant DNA orientation
Why is DNA sequencing useful?
Can check for mutations
Describe the steps involved with getting DNA out of bacteria (mini-prep)
- grow bacteria
- break them open
- plasmids are small and compact
- DNA is big & tangled
- results in purified DNA
What is a restriction digest?
Digest DNA with a restriction enzyme.
What technique is used to separate DNA fragments based on their sizes?
DNA electrophoresis
What gel is used in electrophoresis?
Agarose gel
Why is ethidium bromide used in electrophoresis?
To visualise the DNA fragments under UV light, as it becomes fluorescent.
What is the purpose of a DNA ladder?
DNA ladder is used to estimate size
What charge electrode does the DNA move to?
DNA is negative so it moves towards the positive electrode.
What moves faster through the agarose gel - small or large fragments?
Small fragments move faster, large fragments move slower
What are 3 other uses of PCR?
- modern genetic fingerprinting
- identification of repeat expansions (certain diseases - e.g. Huntington disease)
- identification of genomic rearrangements (cancer)
What is RFLP?
Restriction Fragment Length Polymorphism (RFLP) - this is where the number of repeats affecting the size of the restriction fragments.
What is the specific type of sequencing, which is another phrase for DNA sequencing ?
Sanger sequencing - a.k.a the chain termination method
What 2 things are needed to be known in order for sequencing to occur?
- How long a DNA molecule is (how many base pairs)
- What the last nucleotide is
