Genetic Engineering and Applications

  • Created by: Sarah
  • Created on: 30-04-17 23:01
what happens in RNA inteference?
presence of double stranded RNA, recognized+cleaved by dicer -> give mircroRNAs -> microRNAs form part of RISC-> cleavage of mRNA by slicer -> cleaved mRNA is destroyed->no translation
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what does risc stand for?
RNA induced silencing complex
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where is the protein of interest attached to in yeast-2-hybrid?
DNAbinding domain of transcription factor
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what do you use to find out what proteins regulate a certain protein of interest?
yeast 2 hybrid
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what is a yeast 2 hybrid screen looking at?
looking for proteins that physically interact with the protein of interest
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what happens in a yeast 2 hybrid?
POI on DNA Binding domain, lots of unknown proteins on AD activation domains, construct a POI plasmid+ a fish plasmid (plasmid with unknown pros on ADs) plput in yeast cells, selection in aa deficient media
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why in a yeast 2 hybrid do you put it in an amino acid deficient medium?
only those with both plasmids grow, the fish plasmid (unknown pro on AD) and POI plasmid
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what needs to interact for the gene to be expressed?
protein on BD and protein on AD to ineract
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how are the unknown proteins that bind to the POI identified?
by isolating yeast colonies, isolating plasmid containing cDNA for unknown protein and sequence it
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how do you find out the function of a certain protein?
observe absence by knockdown or knockout
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what comes under target validation?
1) knockdown using siRNA 2) Gene knockout in transgenic mice
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what does siRNA stand for?
short inteferring RNA
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what does the RNA inteference pathway do?
regulate gene expresion using microRNA
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one of the first thing in the microRNA inteference pathway is dicer what does it do?
cleaves double stranded DNA into 21-22 long fragment
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what happens when you experimentally do a siRNA gene knockdown?
foreign double stranded RNA is introduced into cells -> dicer cleaves -> risc degrades target mRNA
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2 methods for transgenic mice?
1) pronuclear injection 2) gene targetting
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in pronuclear injection where is foreign DNA (trangene) introduced into?
nucleus of fertilised ova
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how many copies of the transgene are inserted in pronuclear injection?
several
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where is DNA integrated in pronuclear injection?
at random sites integrated into the genome
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what is pronuclear injection used to generate?
GM crops and animals
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why is each trangenic line in pronuclear injection unique?
each foreign DNA (transgene) can be modulated by the insertion site (variable expression patterns)
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what happens in gene targetting?
ES cells from mouse-> target vector electrocorporation->chose cell with target gene->grow in colony->inject ES cells into host blastocyst->implant in foster mother->give birth to chimeric mice->breed chimeric
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what 2 gametes are produced from chimeric mouse?
normal host blastocyst gametes and modified ES cell gametes
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how do you produce a PrP(protease resistant protein) Knockout mouse?
target the PRNP gene
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what is the difference between the homozygous and heterozygous protease resistant protein knockout mutant?
heterzygous have a longer incubation before onset of the gene
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what did trangenic mice with different levels of expression of prp confirm with pronuclear studies?
incubation time is inversely related to PrP expression
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where is foreign dna introduced into in gene targetting
cultured mouse stem cells
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what is gene targetting used to make?
knockins knockouts
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where in the genome does gene targeting intergrate DNA?
specific sites in the genome
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what does microarrays use to hybiridse to each other?
single stranded DNA
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what happens to non-complementary dna in microarray hybridization?
it hybridises to other probe cells or its washed away
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what does red mean the gene has become in microarrays?
up regulated
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what does a microarray chip contain?
a grid of thousands (45,000) probe cells
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what do microarrays compare?
a transcribed gene
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2 applications of genetic engineering?
forensics science, clinical medicine
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2 reasons for a genetic screen?
1) predictive 2) diagnostic
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what is a genetic screen used to predict? example?
variants of BRCA1 and BRCA2 are associated with variable risk of developing breast cancer
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what do BRCA1 and BRCA2 do in breast and ovarian tissue?
control cell division
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what is a diagnostic example of genetic screen?
detection of invading organisms (PCR to screen blood for HIV) or endogenous genetic mutations (SCA)
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what does REFLP stand for?
Restriction Fragment length polymorphism (variations)
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what does it mean that a mutation may create a polymorphism?
a polymorphism in the number/sizes of DNA fragments produced by a particular restriction enzyme
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what can polymorphisms be useful for?
marker for disease as marker of mutation
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how does a mutatioon lead to a polymorphism?
point mutations can abolish or create a restriction enyzme site
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what restriction fragment length polymorphism detects the sickle cell gene?
Ddel
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the normal beta alle for sickle cell is split into what?
175 bp 201 bp
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what are the advantages to in situ testing of DNA/rna and protein?
tissue distribution, function and changes in either
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what is used to see protein in situ?
immunocytochemistry/immunohystochemistry
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what is used to see DNA in situ?
chromome painting or chromosal spreads
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what is used to see protein homogenates?
western blotting
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what is used to see RNA blotting?
northern blotting
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what are the advantages of looking at homogenates?
quatification, size and isolation
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why are homogenates a disadvantage?
requires larger quantity of tissue
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what is the blotting techniques?
1) size separation by gel 2) transfer out of a gel onto membrane (blotting) with nitrocellulose or nylon filter 3) detection on membrane by labelled probe
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what is the disadvantage of in situ visualisation?
requires tissue processing, limited by reagents and resolution
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how does gel electropheresis work?
DNA is loaded into a well with a pre-cast gel, DNA migrates through the geltowards positive electrode, sorted according to size, smaller move faster
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why does dna move towards positive electrode?
dna is negatively charge as phosphate group
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the gel is a porous matrix that acts like what?
a sieve
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what are the 4 factors that affect DNA migration?
1) DNA size 2) gel concentration 3) gel type 4)DNA shape
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does dna move faster in a concentrated 1.5% gel or a 0.7% gel?
0.7% gel
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what is the order of dna shapes from fast to slow?
supercoiled, linear, circular
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what are the 2 gel types?
agarose gel and polyacrimide gel
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which gel is for 100-20,000 bps?
agarose
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how many bps is polyacrimide used for?
10-700bps
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what is the advantage to polyacrimide gel?
better resolution- separates dna differing in 1bp
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what ways can you transfer dna out of gel to a membrane (blotting)?
1)nitrocellulose or nylon filter 2) capillary action 3) blotting by an electric field+sponges
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what is a probe used in blotting to detect it on a membrane?
p-32 probe
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what is DNA/RNA hybridisation?
specific base pairing of 2 complementary single stranded molecules to form a double stranded molecule
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what does detection of a specific DNA/rna rely on?
hybridisation
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what does the stability of hybridisation depend on?
the degree of match between the target and the probe
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what oes detection of a specific protein in a western blot rely on?
specific antigen-antibody interactions
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what does a western blot require?
primary antibody, secondary antibody substrate to add in then oxizided products give off light
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why is in situ detection of RNA importnat?
all tissues of an organism have exactly the same DNA but transcribes a unique subset to RNA
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what does transcribed RNA give the cell?
its identity
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what does rna in situ hybridisation rely on?
principle of hybridisation of complementary (cDNA) and RNA sequnces
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what in situ hybridisation used for?
1) to detect and quantify mRNA 2) to locate RNA seqs within a tissue
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where is NMDR genes expressed how did they detct them?
in the brain used in situ hybridisation to detect and locate expression
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what does the intensity of signal correspond to in in situ hybridisation?
the level of expression
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how do you do analysis of situ hybridisation?
tissue embedded in block transfer onto film, apply probe to tissue slice wash unbound probe off, detect probe on slice
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what does a proteins location in the cell relate to?
its function
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what does immunhistochemistry/immunocytochemistry relie on?
secidic antigen-antibody interaction
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what is the 3 purposes of DNA sequencing?
1) predict the function of DNA 2) to identify exact differences between normal and mutated 3) to confirm seq of engineered DNA
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what method is used for dna sequencing?
the sanger or chain termination method
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what is the dna to be sequenced used as in vitro?
used as a template for dna synthesis
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what do you need for dna synthesis (sequencing)?
a short primer
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why do you need to know some of the sequence of the dna you want to sequence?
to make a primer
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how does the products of dna synthesis being different sizes betray the template sequence?
normal nucleotides are spiked with terminator nucleotides that are tagged, no more nucleotides added after terminator
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what is the ratio of normal : terminator nucleotides?
100:1
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normal deoxynucleoside triphosphate (dNTP) has what at carbon 3?
OH at carbon 3
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what is ddNTP -dideoxynucleoside trispohosphate (terminator nucleotides) missing?
no OH group at carbon 3
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how is the sequence determined after you have different DNA strands of different lengths in sanger or chain termination?
capillary gel electropheresis, laser and detector by computer
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what does sequencing produce?
a chromatogram
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current advantageous dna sequencing technologies?
deep sequencing =coverage 1x vs 7x, high throughput- whole genome in a day, RNA sequencing = includes mRNA, microRNA
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purpose of restriction enzymes?
1) to enable analysis of dna (southern blotting) 2) to enable genetic engineering(cut and paste)
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what are restriction enzymes?
enzymes that recognise specific sequences of DNA and cleave DNA at these sites
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where are restriction enzymes isolated from?
bacteria
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why do bacteria have restriction enzymes?
to protect them from bacteriohages
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how does the bacteria protect its own DNA from restriction enzymes =?
methylation of its own restriction sites
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what is the name of the restriction enzyme derived from? example?
from the source bacteria. EcoR1 is isolated from Escherichia coli
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what DNA do restricition enzymes cut?
double stranded DNA
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how long are restriction sites usually?
4-8bp
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what is a feature of restriction sites?
palindromic- sequence of antisese is the same as sense from orientation
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what 2 dna ends do restriction enzymes produce?
sticky ends and blunt ends
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what has to ligate to form a new dna molecule in restriction enzymes?
compatible sticky ends (cohesive ends)
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what are 4 base site cutters (every 256 bases)
frequent cutter
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what is every 8 base site cutters once every 65536 bases called?
rare cutters
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what is the polymerase chain reaction?
a method used to amplify dna by repeated cycles of in vitro (outside) DNA replication
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dna amplification proceeds in a what?
exponential scale
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what is the 3 things PCR is used for?
a) detection eg HIV infectious agents and forensics b) amplication of small amount of dna (eg for cloning) c) modification- can add point mutation or restriction site
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what do you need for PCR?
1) small amount of template dna seq to be amplified 2) primers of the start and end of the seq 3) DNA polymerase (Taq) and lots of dNTPs 4) thermal cycler
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why is special dna polymerase taq used?
can work at high temperatures
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where is taq from?
thermophillic bac that lived in extreme conditions near volcanoes
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where is the primer for PCR 5' end primer derived from?
sense strand
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where is the anti sense strand of the 3' end primer from?
antisense strand
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3 steps in pcr?
1) denaturation 2) annealing of primers 3) elongation
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what do PCR products become in the next cycle?
the templates
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why is it possible to insert mods like mutations into PCR?
the primers are incorporated into the product
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what can primers in pcr add to the dna?
restriction sites and mutations
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what would you add restriction sites in y pcr for?
to enable genetic engineering
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what is prodduced by adding restriction sites by pcr?
template with primers anneal, pcr product incorporates primer, cohesive ends can be produced when RE cuts
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what does cloning mean?
1) isolate dna of interest 2) maintain dna of interest outside biological context 3) generate unlimited supply of it
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where is dna insterted into in cloning?
into a host dna molecule- vector
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what is a vector?
a dna molecule that is maintained and replicated naturally by a host organism (baceria) fusing insert to vector gives these properties
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what can be used as a vector?
plasmids
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what are good organisms for DNA cloning?
bacteria and viruses
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why are bac and viruses good host organisms?
short life spans, small so keep loads, easy and cheap to grow large quantities, easy to break up and purify dna from them
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what are plasmids?
small circular DNA (3kbp) molecules found naturally in bac
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what are the essential properties of a cloning plasmid?
1) Ori (origin of dna rep) 2) antiobiotic resistance gene to select for bac 3) restriction enzyme sites to insert DNA
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why do you need an Ori- origin of replication in a cloning plasmid?
so it can live without bac and replicate independently
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first step in dna cloning?
generation of compatible sticky/cohesive ends in both insert and plasmid by the same restriction enzyme
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why is the same restriction enzyme used on insert and plasmid?
sticky ends
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what is the second step in dna cloning?
fusion of insert to plasmid using dna ligase
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what is the 3rd step in dna cloning?
put recombinat DNA (plasmids with the insert) into bac (transformation)
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how do you know which bac took up the plasmids?
replica plating, antibiotic resistance selection
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what is transformation?
recombinant dna into bac
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what is the 4th step of dna cloning?
antibiotic selection for bac containing plasmids
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so how does antibitoic selection for bac containing plasmids work?
bacteria on medium and antibiotic, any without plasmids therefore antibiotic resistance gene die
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what is the last step in dna cloning?
large scale propagation of bacterial colonies to isolate recobinant plasmid
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what 2 types of plasmids are there?
1) cloning plasmids 2) expression plasmids
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what types of expression plasmids are there?
1) bacterial expr plasmids 2) mammilian expression plasmids
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what is true about expression vector but not for cloning plasmid?
an expression vector is a cloning plasmid
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what are dna libraries?
population of identical vectors (plasmids) each containing a different insert
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where can a dna library be kept?
in a tube in solution or plated
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what are dna libraries for?
to have all fna ffrom an organism avaliable for further studies
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what type of DNA library is it when it conatins the complete DNA seq of an organism (mouse or human library)?
genomic library
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what is a cDNA library?
derived from mRNA much more detailed, representpart of the genome made into mRNA. can be derived different organs and in different dev stages
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what does the structure of a mammlian expression plasmid contain?
Ori in mammilian cells, antibiotic resistance , eukaryotic promoter
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what is the antibiotic reisistance in mamilian cells?
neomycin
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what is the antibiotic resistance in bac?
ampicillin
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what is the difference between mammalian and bacterial expression plasmids?
bac expr plasmid contains bacterialpromoters, mammilian expr plamsids- eukaryotic promoters
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what must expression plasmids contain to intiate transcription of the insert?
promoter
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what is the difference between a cloning plasmid and expression plasmid?
hhj
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Card 2

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what does risc stand for?

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RNA induced silencing complex

Card 3

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where is the protein of interest attached to in yeast-2-hybrid?

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Preview of the front of card 3

Card 4

Front

what do you use to find out what proteins regulate a certain protein of interest?

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Preview of the front of card 4

Card 5

Front

what is a yeast 2 hybrid screen looking at?

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