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
what does heat shock do to help transformation?
induced heat shock proteins to repair the membrane after taking up the new DNA
how can you tell if a cell has taken up the new foreign plasmid?
grow it in selective media
if it contains resitance to ampicillin, only collonies which have taken up the plasmid will grow
how do you set up a DNA library
cleave human DNA with restriction enzymes
fragments inserted into plasmids
introduce these into bacteria
culture of the bacteris is known as genomic library and useful for identifying genes
how do you find a gene of interest in the genomic library
use hybridisation
what is used to make the DNA molecules stick to the filter
sodum hydroxide
what is the process of using the genomic libray
- filter paper pressed against master plate transfering cells
- filter treated to denature DNA. ssDNA treated to stick to filter better
- filter laid on photographic paper to expose it
- develop paper. reference makrs show colonies with gene of interest
what technique does DNA sequencing exploit?
dideoxy nucleotides
what is a dideoxy nucleotide
it blocks synthesis so if incorporated into DNA the synthesis is blocked and bonds cannot be formed any more
describe how sequencing works?
- primer bound to template
- 4 reactions: polymerase, each base, one radioactive and dideoxy
- synthesis at end of primer
- if dideoxy is incorporated, synthesis stops ( chain termination)
- varied size fragments all ending in A
- run on gell
- size varies position on gel
- ladder of bands can be read
how are the bands read for sequencing?
bottom to top
now automated
what is the modern way to sequence?
run all in one tube, automated dideoxy is furscent each base new colour laser identifies the groups peaks on graph read as the sequence
what is an intron?
non coding section
space the exons out
what is reverse transciptase?
mature RNA molecule witha poly A tail
functions as a primer to initiate synthesis
what does reverse transcriptase do?
uses RNA as a template to make copy to make DNA
how does reverse transcriptase work?
synthesises 5 to 3
creates a hairpin loop
DNA polymerase then used
double stranded cDNA made
what is the hairpin loop?
used as an imitation for the synthesis of the second strand of cDNA
backround on the human growth hormome
anterior pituitary gland hormone
endocrine
what does the growth hormome do?
promotes proliferation of soft tissue and bone growth in childhood
linear bone growth
what was the issue with harvesting growth horome
purified it from donors
but people developed a disease from the GH
what disease did people develop from taking purified GH?
spongioform enchphalopathies
what is recombinant GH?
newer alternative
GENENTECH: Herbert Boyer
what needed to be overcome during production of recominant GH?
need a source of the GH gene
need a suitable vector to excess it
how did they find a source of the GH gene?
used a cDNA library
how was the cDNA library used to find GH gene
took RNA from pituitary synthesised this into cDNA cloned into plasmid cDNA library used found a clone containing this gene used the gene encoding human placental lactogen as a probe as its similar
what other issues did recombinant GH face?
no convenient cutting sites in the gene as the signal sequence for GH is not part of the mature protein
what is PCR?
a chain reaction using the polyermase to make large amounts of target small DNA to make lots of copies
descrbe the steps of PCR
- heat to 90 degrees
- strands separate
- cool to 55 degrees, primer aneals
- anealing step
- warm to 70 degrees, extention step
- DNA polymerase
- new cycle again to make even more DNA
what was the issue of initial PCR?
no polymerases would work at the high temperates
how did scientists get around polymerase not workin at high temps?
used taq polymerase which can tolerate high temperatures
why cant PCR work indefinitely?
in theory it will but reagents eventually run out and the enzymes become exhausted
will only run for a max of 30-40 cycles
what is a microarray?
uses the principles of base-pairing and hybridiation to identify expression of a gene in a cell/tissue
describe the process of a microarray
- the mapped out grid on the chip contain DNA
- RNA is isolated from the chosen cell/tissue. through reverse transcriptase cDNA is formed and this is radiolabelled
- cDNA is then added to the chip and allowed to hybridise
- if it hybridised, it will fluoresce
- laser machine detects this and looks at patterns
how does the microarray chip work?
- Each spot on a microarray contains multiple identical strands of DNA.
- The DNA sequence on each spot is unique.
- Each spot represents one gene.
- Thousands of spots are arrayed in orderly rows and columns on a solid surface (usually glass).
- The precise location and sequence of each spot is recorded in a computer database.
- Microarrays can be the size of a microscope slide, or even smaller.
when are microarrays useful?
- see what genes are expressed in certain tissues
compare two types of tissues eg cancer and normal
why are eukaryotic genes harder to study?
contain introns
what is cDNA
made by the action of reverse transcriptase and represents a DNA copy of the mature mRNA transcript
