Manipulating genes and genomes

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Who experimented on S.pneumoniae in 1928?
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What do virulent strains do?
Cause pneumonia
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What do avirulent strains do?
Don't cause pneumonia
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What are the characteristics of a virulent strain?
They have a polysaccharide capsule for protection
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Are virulent strains smooth or rough?
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What are the characteristics of an avirulent strain?
They don't have a capsule
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Are avirulent strains smooth or rough?
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What happens to mice injected with heat-killed smooth strains?
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What happens to mice injected with heat-killed smooth strains and rough strains?
They contract pneumonia and die
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What what did griffith discover?
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What experiement did avery and mcCarty do in 1944?
They removed proteins and digested the capsule of smooth bacteria. They were left with a precipitation of fibres that were able to transform
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How was the precipitate of fibres deactivated?
Using deoxyribonuclease
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What what did Avery and McCarty discover?
That the transforming principle was DNA
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What experiment did Hershey and chase do?
They used radio isotopes P32 and S35 in order to follow the movement of proteins and DNA when the bacteriophage T2 infected E.coli
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Why was radioactive phosphorus used?
Because only DNA contains it
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Why was radioactive sulphur used?
Because only proteins contain it
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How were bacteriophages made so that they contained the radioisotope elements?
They were allowed to infect E.coli cells that were grown on a medium contain the radioisotopes
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What was the result of the experiment?
Labelled DNA was transferred into E.coli cells whilst labeled proteins were not
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What are the two purines?
Adenine and Guanine
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Are purines double rings or single rings?
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What are the two pyrimidines (DNA and RNA)?
Cytidine and Thymine in DNA, Cytidine and Uridine in RNA
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Are pyrimidines double rings or single rings?
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What is different between deoxyribose and ribose?
Deoxyribose has 1 OH group, ribose has 2 OH groups
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What bonds join nucleotides?
A phosphodiester bond
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At what end does DNA elongate?
The 3' end
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Who proposed the antiparallel model?
Watson and Crick
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Which way does DNA twist in?
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What is the diameter of the major groove?
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What is the diameter of the minor groove?
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What is the diameter of DNA?
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What is double stranded DNA wrapped around?
A nucleosome made up of histone proteins
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Who proposed the idea of semi-conservative replication?
Meselson and Stahl
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What is semi conservative replication?
Each newly synthesised strand of DNA contains one strand of parental DNA and one strand of new DNA
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Which prokaryote RNA polymerase is use for DNA replication?
RNA polymerase III
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What is 3'-5' exonuclease?
It enables RNA polymerase to remove nucleotides at the 3' end so if the wrong nucleotide is added it can be removed
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What is 5'-3' exonuclease activity?
It enables RNA polymerase ro remove sequences that have already been synthesised
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Which way does the leading strand run?
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Which way does the lagging strand run?
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What does DNA helicase do?
Unwinds the double stranded helix
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What do single-stranded binding proteins do?
Stabalize ** DNS
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What does DNA gyrase do?
It relieves tension in the DNA helix that is caused by DNA unwinding
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How is DNA replicated on the lagging strand?
The strand forms a loop so that nucleotide polymerisation can happen in the 5'-3' direction. After about 2,000 bases are added an okazaki fragment with be reached and DNA polymerase releases the strand
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What is an okazaki fragment?
Fragments of DNA produced during DNA replication formed by DNA primes which binds complementary bases upstream of DNA polymerase
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What dos DNA ligase do?
It joins the phosphodiester backbone
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When does termination of DNA replication occur?
At defined terminator sequences
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What happens in G1 of the cell cycle?
Transcription and translation of cell proteins
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What happens in S of the cell cycle?
DNA synthesis occurs
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What happens in G2 of the cell cycle?
Its a second growth phase
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What are the steps in mitosis?
Prophase, metaphase, anaphase, telophase
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What is true about transcriptional activity in prokaryotes?
Most protein-coding genes are transcriptional active by default
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How is transcription controlled?
Its maintained by repressor proteins
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Where do repressor proteins bind?
To DNA sequences adjacent to the RNA polymerase binding site
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What does binding of a repressor protein do?
It prevents RNA polymerase from binding
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What bacteria is the lac operon found in?
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What does the lac operon control?
The expression of genes involved in degrading lactose in order for it to be used as an energy source
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What does the Pi gene encode?
The promotor for the Lac i gene
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What does the Lac i gene encode?
The lac repressor protein
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What is the CAP site?
The binding site for cAMP
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What is the P site?
The binding site for RNA polymerase
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What is the O site?
The binding site for the repressor protein
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What does the LacZ gene encode?
B- galactosidase
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What does B- galactosidase do?
It breaks lactose into galactose and glucose
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What does the LacY gene encode?
Lactose permease
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What does lactose premise do?
Its an enzyme which brings lactose into the cell for it to be hydrolysed
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What effects does an absence of lactose have on the lac operon?
The RNA polymerase is bound to the promoter site but it cannot transcribe genes because the lac repressor is bound to the operator site
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What effects does the presence of lactose have on the lac operon?
Lactose binds to the repressor protein causing it to fall off on the DNA, RNA polymerase is able to move along the DNA and transcribe the genes
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How is lactose able to get into the cell in the first place?
Lac genes are transcribed at very low levels even when lactose isn't present
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What effects does the presence of glucose have on the lac operon?
cAMP production is inhibited and transcription of the lac genes is suppressed because cAMP in needed for transcription
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What happens if the glucose concentration is low?
cAMP production is high. cAMP binds to the CAP site which activates the transcription of structural genes
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What is catabolite repression?
Its the mechanism by which glucose can inhibit transcription of the lac genes by not allowing full activation
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What are the two ways that transcription can be terminated?
rho-dependant and rho-indépendant termination
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What is rho-dependant termination?
When the rho protein binds to the newly synthesised mRNA and causes termination when RNa polymerase pauses at certain points
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What is rho-independant termination?
It involves intrinsic sequences that form hairpin loops in the RNS and force RNA polymerase to dissociate from the RNA
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Which eukaryote RNA polymerase transcribed mRNA?
RNA polymerase II
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What is needed for RNA polymerase to bind to DNA?
Transcription factors
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How is DNA transcriptions signalled?
Activators bind to DNA which message for recruitment of modifying proteins to remove histones from the DNA promotor site
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What is the structure of mature mRNA
It has a methyl cap at the 5' end and a poly A tail at the 3' end
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What is an intron donor site?
The start of an intron
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What sequence on the mRNA signals a donor site?
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What is an intron acceptor site?
The end of an intron
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What sequence on the mRNA signals an acceptor site?
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What i used for splicing?
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What genes can be affected depending on the sex of drosophila?
sxl/ tra/ ds.
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What do restriction enzymes do?
Cleave DNA at particular sequences within a DNA sequence
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What restriction enzyme recognises GAATTC?
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How does the E.coli strain that produces EcoR1 protect itself from DNA cleavage?
They produce a specific methylase which attaches methyl groups to the EcoR1 sites to prevent cleavage
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What types of restriction enzymes recognise DNA sequences that are symmetrical?
Type II
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What are palindromic sequences?
Double stranded DNA that reads the same sequence forwards and backwards
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What does DNA ligase require to bond the phosphodiester backbone?
A free -OH group and ATP (animals) / NAD+ (bacteria)
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What is the optimum temperature of DNA ligase?
37 degrees
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What is DNA cloning?
The ability to extract a fragment of DNA from one source and insert it into the DNA of another
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What is a vector?
A closed circular double-stranded DNA plasmid molecule
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What is involved in chemical transformation of DNA?
E.coli is suspended in a solution of cacl2 which forms holes in the bacteria. Plasmid DNA is added and a heat-shock initiates the movement of DNA into the cell
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What is involved in electroporation of DNA?
It uses an electric field pulse which induces pores into the membrane of E.coli. Plasmid DNA can then enter the cell before the pore recluses
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What is involved in using a gene gun DNA transformation?
The plasmid DNA is coated in a microscopic bead which is then loaded into a firing chamber of the gene gun and a force expels the beads. The bead stops whilst the DNA continue to move into the target cell
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How do you isolate plasmid DNA from bacterial cells?
Bacteria are treated with a detergent SDS to disrupt the membrane and denature cell proteins and DNA. Potassium acetate is added to aggregate the degraded cell parts
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How is plasmid DNA purified?
using an exchange column
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In gel electrophorus what DNA fragments move the furthest?
The smallest one
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What is southern blotting used for?
To radioactively label DNA that has been separated by gel electrophoresis with a probe. Allows you to detect DNA sequences that are complementary to the probe
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Whats northern blotting used for?
Detecting RNA sequences
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Whats western blotting used for?
Detecting proteins by using an antibody against them
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What was the first cloning vector?
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Wha features are present in a PUC plasmid vector?
A mutated colE1 ori, ampicillin resisitance gene, LacZ gene
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Why is the LacZ gene used?
It encodes an a-protein which contains multiple recognition sites for multiple enzymes. This DNA sequence is used to insert foreign DNA
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What is used in blue-white screening of the PUC plasmid?
Xgal and IPTG (b-galactosidase inducer)
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What would a blue result show?
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What would a white result show?
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How is phage DNA circularised?
cohesive ends (COS)
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What is the outcome of the lytic pathway?
The host cell dies and phage particles are made and released
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What is the outcome of the lysogenic pathway?
The phage genome is integrated into the cells genome and phage DNA is replicated everytime the bacterial genome is
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What pathway is taken when CII is active?
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What pathway is taken when CI is active?
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What does active CII do?
It activate the repressor of CI
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In a good growth medium is CII active or not?
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Because poetess are made in a good growth medium and they breakdown CII
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What are insertional Vectors?
When insert DNA is cloned in-between two restriction enzyme sites
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What are replacement Vectors?
When insert DNA replaces a piece of the vector DNA
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What type of bacteria for M13 phages infect?
F pillus bacteria
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What type of genome does M13 pages have?
A ** genome
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What is unique about M13 vectors?
They don't kill the host cell, only restrict its growth
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How do bacterial cells infected with M13 look?
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What are cosmic vectors?
Plasmids that contain the phage cos site
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Why are cos sites needed?
Because they are the only requirement for in vitro packaging in phages
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What are the different types of artificial chromosomes?
Yeast AC (YACs) , P1 AC (PACs), Bacterial AC (PACs), Human AC (HACs)
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What is PCR?
The repetitive copying of a selection of double-stranded DNA
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What are the two primers needed for RNA polymerase?
A primer that same as the sense strand and the antisense strand
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What happens in the denaturing step?
DS DNA is separated by heating to 94 degrees
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What happens in the annealing step?
The two strands are cooled in the presence of the two primers. They bind complementary so that the two -OH groups are facing each other
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What happens in the extension step?
DNA polymerase is added to the 3' end of oligonucleotides. It synthesises new DNA using oligonucleotides
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What DNA polymerase is used?
Taq polymerase
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Why is it used?
Because it can withstand high temperatures used in PCR
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What does Taq polymerase lack?
3'-5' exonuclease activity
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What is reverse transcriptase PCR (RT-PCR)?
A method of RNA amplification and the ability to quantify it from DNA product
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What happens in RT-PCR?
RT reads the mRNA strand to produce a ** DNA molecule, The cDNA can then be synthesised into a double stranded molecule
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What is real-time PCR?
Its an automated version of RT-PCR and quantifies reaction products for each sample in every cycle
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What does it use to quantify product?
A fluorescent reporter- the amount of signal is proportional to the amount of PCR product
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What do genomic libraries contain?
Representative copies of all of the genetic material of an individual organism
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What type of DNA do they contain?
Genes expressed and not expresses, introns, exons, promoters and terminators
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How can genomic DNA be cleaved?
By mechanical shearing or restriction enzyme digestion
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What is involved in mechanical shearing?
DNA is passed though a narrow-gauge needle to break it up
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What is involved in restriction enzyme digestion?
Uses restiction enzymes to cut DNA at restriction enzyme sites
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What is partial digestion?
A process where parts of the genome are cut at different times so that they can be sequenced overtime and to identify overlapping sequences
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How are cDNA libraries made?
Through the conversion of mRNA into DNA fragments so that they can be cloned into vectors
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What do cDNA libraries contain?
Only coding regions of the genes expressed in sample taken from specific tissue
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What can cDNA libraries reflect?
The relative abundance of mRNA in the original tissue
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Why can't the mRNA be cloned directly into vectors?
It is too unstable do a DNA copy is needed
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What is used to create a cDNA library?
reverse transcriptase
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What does directional cloning use?
Modified oligonucleotides so that cloning of cDNA fragments can only happen in one direction
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What are subtraction libraries?
cDNA libraries made by removing the common cDNA sequences from two samples so that you are left with only unique sequences to that tissue
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How can a gene be identified by looking at the DNA sequence of the clone?
Nucleic acid hybridisation
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What is nucleic acid hybridisation?
Use a radioactive probes that is similar to the DNA sequence. DNAmust de denatured into ** DNA and probes bind at the point of the target gene
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How can a gene be identified by looking at the protein sequenexpressed by a gene?
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How is immunoscreening done?
The cDNA fragment is cloned into an expression vector (lambda zap) under control of the lac promotor. They are transformed into bacteria in lactose. The gene is expressed
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What are the two examples of immortal cells?
HeLa and CHO
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What is a transient transfection?
When the foreign DNA is transfected into the cells but not integrated into the genome
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What is stable transfection?
When the DNA is transfected into the cell and integrated into a random location of the genome
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Wat is involved in chemical transfection?
Cells are washed in a phosphate buffer, DNA and cacl2 is added. The precipitate formed is internalised into the cell
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What type of virus is a SV40 virus based on?
Simian virus 40
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What are the early genes in SV40?
Genes that are expressed immediately following infection and are required for viral replication
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What are the late genes in SV40?
Genes expressed later on during the infective cycle
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What genes do adeno-viruses late?
E1 ad E3 genes
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What does that prevent the viruses from doing?
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How is the issue over come?
They are propagated into cells have have the missing genes present
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Is adeno-virus infection transient or non-transient?
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What family of viruses are AAV viruses?
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What do they require to replicate?
Adeno virus or environmental stimuli e.g UV light
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What happens to an AAV if an aden-virus isn't present?
The genome integrates into the cells genome at chromosome 19 and remains an ineffective virus
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What do AAV vectors require?
2 ITR sequences flanking the insert DNA
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What are the 2 types of retroviruses?
HTLV and Lentiviruses
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The structural genes of retroviruses are what?
gag, pol and env
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What does gag encode?
The capsid proteins
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What does pol encode?
reverse transcriptase
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What does env encode?
The surface and transmembrane proteins
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What is pronuclear injection?
When DNA is injected directly into the pronuclei of a freshly fertilised embryo
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What genetic make up will the pup have if hey have integrated the DNA?
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What are the 3 classes of knock-out mice?
Lethal, observable phenotype and no phenotype
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Give an example of a lethal knock out
Deletion of hsp47
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Give an example of an observable phenotype in knock out mice
Deletion of tumour suppressor P53
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What is nuclear transfer?
When the nucleus of a differentiated cell is injected into an enucleated oocyte
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How can you measure ongoing transcription?
A nuclear run-on assay
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What does a nuclear-run on assay involve?
Stopping transcription, putting cells into a buffer contain radioactive UTP and dNTPs. Transcription is reactivated and new mRNA can be identified
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What is used to measure protein levels?
Immunoblots ( western blotting)
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How can you measure the steady state of mRNA?
Microarrays and PCR
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What does an affymetrix chip contain?
Chemically synthesised single-stranded oligonucleotides
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What is the chip acting as?
A template for the binding of cDNA
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What colour is cy3?
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What colour is cy5?
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What does chromatin immunoprecipitation do?
It identifies DNA binding sites for particular transcription factors
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What is the aim of the technique?
To identify which genes are controlled by specific transcription factors
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What creates the cross links between proteins and DNA?
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How is the DNA sheared?
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How can you find specific proteins?
Create an antibody against it
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What does binding of mRNA to antisense RNA do?
Interfere with translation of the mRNA so the protein is made at a much lower level
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What is RNA interference?
The introduction of double stranded RNA molecules into cells which reduces gene expression
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In drosophila, introducing which gene induces cell cycle arrest?
Cyclin E gene
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When extra copies of a gene are introduced into plants what happens?
Both the introduced and original genes are switched off
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What enzyme breaks down ds DNA?
The dicer
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What fragments are produced by the dicer?
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What do siRNAs bind to?
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What does RISC also bind?
The mRNA molecule that is complementary to the siren
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What does RISC do?
It cleaves the bound mRNA
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Why do plants undergo co-suppression?
It is a defence mechanism against RNA viruses that replicate via a double-stranded intermediate
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Card 2


What do virulent strains do?


Cause pneumonia

Card 3


What do avirulent strains do?


Preview of the front of card 3

Card 4


What are the characteristics of a virulent strain?


Preview of the front of card 4

Card 5


Are virulent strains smooth or rough?


Preview of the front of card 5
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