Manipulating Genomes Flashcards
How does Gel Electrophoresis work to separate DNA ?
- fluorescently label DNA
- DNA fragment inserted at negative electrode of gel
- DNA negatively charged due to phosphate groups so it moves to anode (+ve electrode) when a current is passed through gel
- smaller fragments move more quickly + further distance
- can identify diff DNA fragments by shining under UV light
How is electrophoresis equipment set up ?
- agarose gel is solidified + placed into tray
- buffer solution added over gel
- both ends of gel connected to a battery + current passed which moves DNA
what is DNA microarray ?
way of assessing an individual’s DNA for the presence
of a specific DNA sequence
what 5 things are needed for DNA sequencing ?
- free nucleotides in excess
- DNA primer
- DNA polymerase
- single stranded DNA to be sequenced
- fluorescently labelled terminator bases
How is the sequence of original DNA read from the gel ?
- read gel from BOTTOM TO TOP = this is complementary sequence
are short strands near bottom or top of gel ?
bottom because they travel furthest distance
Describe the process of DNA profiling (6) ?
- extract DNA
- amplify DNA sample using PCR
- restriction endonuclease cuts DNA at specific restriction sites
- carry out gel electrophoresis
- southern blotting using a nylon membrane
- hybridisation = add radioactive / fluorescent DNA PROBE
3 Stages of PCR + what machine does it go in ?
- denaturation (95 deg)
- annealing (55)
- extension (72)
- in a thermocycler
what happens during denaturation ?
- DNA heated to 95 c
- H bonds broken bet complementary bases
- 2 single strands of DNA created
what happens during annealing ?
- cooled to 55 c
- primers annealed to single stranded DNA by H bonds
what happens during extension stage of PCR ?
- temp raised to 72
- free nucleotides line up against exposed bases via complementary base pairing
- Taq polymerase forms phosphodiester bonds
what is difference bet somatic gene therapy + germline therapy ?
- somatic = targets cells within affected organ only eg. lungs, so offspring could still inherit diseases
- germline = targets sex cells so offspring cannot inherit illness + currently illegal in humans
Explain why only selected sections of non-coding DNA are used when profiling a human (3)
- most of human genome is same
- so using coding sequences do not produce unique sequences
- non coding DNA has repeating sequences
Describe three differences between the process of thin layer chromatography and the form of electrophoresis used to sequence DNA (3)
- tlc seperated by adsorption vs ec = size #
- no buffer in tlc
- no current in tlc
Explain why is the DNA of prokaryotes easier to sequence than that of eukaryotes.
- no introns
- no histone proteins
- non non-coding sections of DNA
