genetic manipulation Flashcards
what issues are there with genetic manipulation?
need to be able to:
purify DNA in large amounts
manipluate in the lab
produce large amounts to study
why isDNA hard to purify>
DNA is chemically homogenous
bases are sugars are repeating units
what is the process for isolating DNA?
- homogenuse DNA using detergent and a blender
- using cel lysate
- use resin with negative charge
- ion exchange chomatography
- wash away other bits no DNA
- salt disrupts charge to get off DNA
- purify this using alcohol precipitation
- centrifge to get pellet of NDA
- disolve in aqueus buffer
what does DNA manipulation mean?
mechanisms for cutting and joining DNA molecules
who discovered plasmids and restriction enzymes?
Boyer and Cohern
what are they key discoveries for DNA manipulation?
observations that phage are host specific and infected dna degrades fast
how many types of restriction enzyme are there?
3
which type of restriction enyme is the most important and why?
type 2
they recognise palindromic sequence and cleave within it
very specifc
how can a restriction enzyme cut?
symmetrically/ blunt ends
asymmetric/ sticky cohesive ends
how many times will a 4 base pair enzyme cut?
256
how many times will a 6 base pair enzyme cut?
4096
how are DNA molecules joined back together after being cut?
enzymes made by bacteria
they can join the nick back together
called ligase
how does ligase work?
the presence of ATP and magnesium will join phosphate with hydroxyl group again
enzye covalently links the two chains and helps with the backbone
how developed agarose gel electrophoresis?
Borst
how does gel electrophoriesis work?
uses an electric charge or separate out charged molecules
the DNA moves through small pores according to ites weight
how does DNA move through agaroe gell?
small pores
small DNA will move faster than large DNA
what effect does super coiling have on DNA and electrophorisis?
makes it heavier and move slower
how are the molecules seen within the gel?
mix a dye into the gel
add ethidium bromide to DNA to bind, this will fluoresce under UV light
how can you prouce large amounts of DNA
attach it to a replicon
what is a replicon?
a piece of DNA with the ability to replicate exisiting in the form of a plasmid
method of obtaining large amounts of DNA
restriction enzymes used to insert the fragment
plasmid digested with restriction enzymes as is the gene of interest
insert/stick together
recombinant molecule made
foreign plasmid to be taken up by cell in transformation.
describe the process of transformation
treat bacterial cells with calcium chloride, make them cold, then heat shock
how does calcium chloride help with transformation
helps them take up the DNA from the environment
lipid layer is negative to DNA cant get though but calcium chloride neutralises this