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113 Cards in this Set
- Front
- Back
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proteome
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proteins expressed by genome
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Explain using lactate and NAD+ how an assay works
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assay is biochemical test which searches for unique property of a protein.
measures the enzyme activity of lactate dehydrogenase lactate + NAD+--ladehy--->pyruvate + NADH + H+ Test looks for absorbtion of light that NADH has that NAD does not. (340 nm in one minute)) |
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What are the two things you need for purifying protein?
Once you have them, what can you measure? |
protein assay to measure enzyme activity
protein concentration with these two measure specific activity |
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specific activity
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ratio of enzyme activity to amount of protein in enzyme
the idea is to maximize and increase the specific activity through purification |
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homogenate
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source of protein from cells and tissues
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differential centrifugation
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homogenizer disrupts cells
homogenate is centrifuged in step by step process yeilding: 500 x g for 10 min = nuclear fraction 10,000 g's for 20 min = mitochondrial fraction 100,000 gs for 1 hour = microsomal fraction |
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Describe salting out
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increased salt decreases a protein's solubility
there are different concentrations for different proteins i.e. 0.8 M ammonium sulfate for fibrinogen 2.4 M ammonium sulfate for serum albumin concentrates dilute protein solutions |
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Dialysis
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separates proteins from small molecules and salts, but not other proteins
can remove ammonium sulfate from protein preparation cellulose membrane is semipermeable |
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what are nucleic acids comprised of?
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DNA and RNA
individual monomers are a sugar, base and phosphate DNA uses 2' deoxyribose for sugar RNA uses ribose the linear polymer is linked via phosphodiester bond from 3' end of one monomer to 5' start of next monomer |
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Base pairings?
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Adenine and Thymine
Adenine and Uracil (RNA) Guanine and Cytosine |
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Which bases are from purine?
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Adenine and Guanine
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Which bases are from pyrimidine?
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Thymine and Cytosine
Uricil |
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What is a nucleoside?
What are the names of the nucleosides for RNA? DNA? |
base and a sugar with a B-glycosidic linkage
RNA--adenosine, guanosine, cytidine, uridine DNA--deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine |
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where does the b-glyocsidic linkage occur with A & G (purine base)?
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N--9
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where does the B glycosidic linkage occur in C, T and U (pyrimidine bases)?
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N--1
all bases attach to C-1 of sugar. |
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why is a nucleotide different from a nucleoside?
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nucleotides include the Phosphodiester linkage
Pi attaches to 5' of sugar |
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what is ATP?
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Adenosine Tri Phosphate
nucleotide |
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What is 3' dGMP?
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deoxyguanosine 3' monophosphate
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What's the nucleotide name for Adenine for RNA and DNA?
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RNA- Adenylate
DNA - deoxyadenylate |
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what's the nucleotide name for DNA and RNA for Guanine?
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RNA -- guanylate
DNA -- deoxyguanylate |
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What's the nucleotide name for Thymine/ uracil for RNA and DNA?
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RNA - uridylate
DNA- thymidylate |
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WHat's the name for nucleotide for cytosine RNA and DNA?
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RNA-- cytidylate
DNA--deoxycytidylate |
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Describe the composition of DNA
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comprised of the 4 nucleotides
deoxyguanylate, deoxyadenylate, deoxycytidylate thymidylate 5'-pApCpGpT-3' chain polarity and only one direction. |
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E coli DNA
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single strand 4.6 mil nucleotides long
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Describe a double helix
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2 oppositely oriented helical polynucleotide chains coiled around common axis
bases are inside, sugars are outside bases perpindicular to axis rise of 3.4 A structures repeat every 34 A so 10 bases / turn and rotation of 36 degrees / base diameter 20A |
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How are bases paired (DNA)?
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G=C
A=T held together by weak H bonds 1-5 kcal/mol van der waals forces = 0.5-2.0 kcal / mol bases are hydrophobic and clustered inside while polar sugars (- charge) are exposed to water |
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What is semiconservative replication?
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basically shows that the double helix splits open and replicates.
In the daughter generation one strand on new helix is from parent and one is nacent |
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Explain the Meselson and Stahl experiment
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E Coli parent DNA labeled with 15N.
Then they grew Ecoli in 14N environment. Showed the incorporation of 14N during replication They could detect the difference due to the density difference (1.66 vs. 1.76 g/mol) after one replication, density was half for 14N and half for 15N after 2 generations equal amounts of hybrid and 14N DNA |
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What is DNA melting?
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DNA helix can be reversibly melted
heat to Tm (temp at which 50% of structure is lost)~70 degrees C or add alkali or acid to ionize bases |
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What is hypochromism?
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a way to determine if bases are stacked or not
Stacked bases absorb less light than unstacked so at Tm single strands will abosorb more uv light at 260 nm |
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What are some characteristics of circular DNA?
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continuous
may be supercoiled to superhelix for compactibility may be relaxed |
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Describe a stem loop structure
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made by ssDNA or ssRNA
may form watson-crick base pairs with other mismatched base pairs can have non-standard base pairing |
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What is DNA polymerase?
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Kornberg discovered it catalyzes DNA synthesis
adds deoxyribonucleotides to preexisting DNA template directed by the existing DNA template some remove mismatched pairings |
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What does DNA replication require to take place?
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DNA n
dNTP (of all 4 bases) 5' deoxynucleoside triphosphates Mg2+ free 3'OH primer |
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How does the template direct DNA replication?
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goes in a 5' -----> 3' direction
3' OH mounts a Nucleophilic attack on the alpha triphosphate chain attached to the 5' of the previous nucleotide PPi (pyrophosphate) is given off |
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describe the order in which a protein is made.
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DNA transcription to mRNA
mRNA takes it to ribosome RNA translates with help from tRNA the codons into proteins |
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Describe mRNA (E.coli)
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template with codons which serves as the master for translation.
1.2 kb long average for E coli 5% of total RNA (ribosomal RNA, tRNA make up rest) |
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Describe tRNA
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one for each of 20 amino acids
~75 nucleotides ~25 kDa carries the activated amino acid and with its corresponding anticodon aids in translation in ribosome makes up about 15% of RNA |
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Describe rRNA
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ribosomal RNA
makes up 80% in E. coli This 80% is comprised of 23S, 16S and 5S species |
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Describe snRNA
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only in Eukaryotes
works in splicing out the inons |
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Describe miRNA
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micro RNA
class of small non-coding RNA's (~21 nucleotides These guys bind complementary mRNAs and therefore inhibit translation |
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describe small interfering RNA
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siRNA
small non-coding RNA bind up mRNA and degrade it |
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Describe RNA polymerases
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catalyzes transcription
uses dsDNA or ssDNA as template makes and elongates RNA chains |
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What does an RNA polymerase need to start elongation?
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ATP, GTP, UTP, CTP
Mg2+ or Mn2+ template but NO primer needed |
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Describe transcription
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RNA polymerase aids for the free 3'OH to mount nucleophilic attack on the incoming alpha phosphate
reaction is aided by hydrolysis of PPi No proofreading here. The new mRNA strand is identical to the coding DNA strand. (not identical to template, but complementary to template) |
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How do mRNA in Eukaryotes differ from those in Prokaryotes?
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There is a 5' cap and a 3' poly AAAAAAAA structure added after transcription
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Describe the role and mechanism of tRNA
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brings amino acids to ribosome for making proteins
aminoacyl tRNA synthetase uses ATP to make aminoacyl-tRNA which is a unique tRNA for each amino acid each one of these aminoacyl-tRNA's have a unique anticoding site which will recognize the codon on mRNA |
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what is and what is the significance of a degenerate genetic code?
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amino acids can have more than one codon.
64 possible codons for 20 amino acids 3 triplets are stop codons so the leftover 61 code for the 20 amino acids degenerate code decreases harmful mutations and having too many chain terminators |
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Where is the amino acid attached to tRNA?
What's on the other end |
3' end of tRNA attaches to the carboxyl carbon of the amino acid
5' end is phosphorolated |
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Which two amino acids only have one codon?
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Trp
Met |
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Which three amino acids have 6 codons?
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Leu
Ser Arg |
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What are the stop codons?
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UAA
UAG UGA |
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What is the codon for Trp
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UGG
Take a Trp to australia to get some uggs |
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What is the codon for Met?
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AUG
Mett's birthday is in AUG |
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XY___ and XY____ ALWAYS encode for the same amino acid
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C and U
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XY____ and XY_____ USUALLY code for same amino acid
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G and A
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For E. coli what starts translation?
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On the mRNA chain a 5' shine dalgano (purine rich) marker occurs at -10, and then an AUG or GUG is +1 which tells the fMet to bind there and begin
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For eukaryotes what starts translation?
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At the 5' cap, Met-tRNA binds to the first AUG it sees and goes from there.
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How does translation know when to stop?
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UAA, UAG and UGA are stop codons
This binds protein which is not tRNA and it uses release factors to release the new protein |
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Describe how universal the genetic code is.
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Nearly universal but some deviations
in humans UGA is a code for Trp and not a stop codon instead we have AGA and AGG as stop codons and we don't code for Arg mitochondrial DNA is different because they make their own tRNA's 16 organisms deviate from the standard genetic code |
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what are introns and exons
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introns are unused code and exons are expressed genes which means we must splice these guys.
Humans have 8 introns which can be 50-10000 nucleotides long |
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Describe human hemoglobin B chain with regard to introns and exons
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3 exons and 2 introns
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Describe how we know about introns and exons?
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pre m RNA is heavier--has a 15S rate as opposed to the actual mRNA which is 9S rate.
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What is a spliceosome?
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assemblies of proteins and small RNA molecules
recognizes that introns begin with GU and end with AG preceeded by essential pyrimidine tract |
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What would be the benefit of exon shuffling or alternative splicing?
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shuffling allows for recombined genes which may provide diversity of function
alternative splicing may lead to new proteins being formed |
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How do restriction enzymes provide the bases for gene exploration?
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restriction endonucleases cut DNA in certain spots where they recognize specific sequences and cut both strands
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Where do restriction enzymes know where to cut?
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the cleavage site is 4-8 bp at a palindromic symmetry site.
They see this symmetry and hydrolyze the phosphodiester bonds here can leave staggered (sticky) or even (blunt) cuts. |
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EcoRI
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5' G*AATTC 3'
3' CTTAA*G 3' derived from E. coli |
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What are the endogenous and laboratory uses for restriction endonucleases
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In cells, these guys cleave foriegn DNA without harming host DNA
In labs, we use it to cleave DNA into fingerprints so we can work with it better |
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Describe SV40
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5.1 kb circular DNA
has 1 EcoRI cleavage sit has 4 HpaI has 11 HindIII |
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Once you have DNA fragments what do you do with them to manipulate them?
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Gel electrophoresis will separate up to 1,000bp
(more porous agarose up to 20 kb) Pulse Field Gel Elect. separates chromosomes |
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what does ethidium bromide do?
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stains dsDNA fragments after separated by gel elect. and has sensitivity to show 50ng with orange fluorescence
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Explain how to identify restriction fragments using 32P probe
which technique is this? |
Separate different fragments in gel
denature them to make ssDNA use a nitrocellulose sheet a strand, matching the one you are looking for has 32P incorporated. when it finds its ppair it links up and audioradiography will show you which one it is on paper southern blotting |
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What other things can southern blots reveal about a restriction fragment?
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mutations on restriction fragment
grounds for polymorphism RFLP restriction fragment length polymorphism |
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What is northern blotting?
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same technique as southern but for RNA
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Describe dideoxy method for DNA sequencing
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The method of incorporating a small amount of dideoxynucleoside triphosphatees which serve to effectively terminate replication at various points
primed with synthesized fragment use DNA polymerase add all the deoxyribonucleoside triphosphates and 2' 3' dideoxy gel and audioradiogram to sequence |
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what's a more modern less radioactive sanger method?
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fluorescence tagging
can run all four bases in one reaction up to 500 bases can be determined automated. can generate more than 1 million bases / day |
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What method is used for making DNA or DNA probes?
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solid phase phosphite triester method
use glass bead add excess reagents to drive rxn to completion sequential addition of protonated (proteceted) deoxyribonucleoside 3' phosphoramidites wash soluable protectors away |
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what are the reagents for phosphite triester method?
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deoxynucleoside 3' phosphoramidite
DMT dimethoxytrityl BCE B cyanoethyl I2 (oxidizer) dicholoroacetic acid NH3 |
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What is the role of NH3 in phosphite triester method?
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remove all protecting groups and remove the deoxyribonucleoside 3' phosphoramidite from the glass bead
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what is role of dichloroacetic acid in phosphite triester method?
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removes DMT from 5' end of newly added deoxyribonucleoside.
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where does the triester form in phosphite diester method on activated monomer and chain?
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phosphit on 3' of activated monomer attaches to 5 ' OH of chain.
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What's the efficiency of the phosphite triester method?
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99%
10 min per cycle elongation is never 100% so the longest one is the golden one. You can find this using PAGE or HPLC |
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Once you makes some DNA using the phosphite triester method, what can you do with it?
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make probes (32P or fluorescence tag and then set it loose in fragments to find the needles in the haystack)
use it as a primer for new neighboring DNA make new designer genes which can make new funcitons or new proteins |
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What is PCR?
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Polymerase Chain Reaction
amplifies your sample |
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What are the 3 steps in PCR>
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strand separation (heat)
hybridize primers (cool with a ton of primers in there --20-30 nucleotides long) heat to 72 so Taq polymerase can start making the strand 5' -3' repeat 25-30 times n cycles = 2tothe n results |
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Describe recombinant DNA technology
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DNA fragment + a vector (plasmids or lambda phage)
host (E. coli) |
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Describe how Eco RI works with plasmid pSC101
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cuts pSC101 at a specific site leaving a sticky end.
If you use Eco RI to cut any piece of inserting DNA those same sticky ends will line up and voila. |
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What is DNA ligase?
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DNA ligase catalyzes the formation of phosphodiester bonds
needs a free 3'OH and 5' phosphate as well as ATP or NAD+ |
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Describe the cohesive ends (Linkers) method.
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If you have a DNA segment you can add cohesive ends to put the tape on the ends.
polynucleotide kinase phosphorylates the 5' end of a linker T4phage ligase seals them (can be blunt ended). then use EcoRI to make the sticky ends and insert into your vector |
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what is a polylinker?
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a synthetic DNA fragment programed with several sequences that many restriction endonucleases recognize. Can custom make your sticky ends.
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why are plasmids and lambda phages good vectors?
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they get the recombinant DNA into bacteria easily which you can then select for.
Plasmids carry genes for inactivation of antibiotics and can replicate independently of host genome |
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What is it about pBR322 that makes it good for cloning?
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has genes for resistance to tetracycline and ampicilin
the EcoRI site isn't in these genes for the ab resistance but it does have other restriction enzyme sites which will interrupt the resistance |
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Describe selection using pBR322
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pBR322 uses the enzyme cut in amp. resistant element and foreign DNA ligated in.
Now these guys are going to be amp sensitive. Still resistant to tetra. grow E coli on plates of tetra. transferred in matching postions to new plates . One plate has tetra and another has amp and tetra. Now you will know which ones are amp sensitive |
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What size is a plasmid?
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2-100+ kb
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What is the size of lambda phage?
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48 kb
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What are the differences between a lytic and lysogenic pathway?
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lytic kills host cell quickly and produces ~100 virons
lysogenic inserts DNA into host cell genome and it is replicated for many generations Upon activation it expresses dormant viral DNA |
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What is lambda gt-lambdaB
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mutant lambda phage with only 2 EcoRI sites as opposed to the normal 5
enter bacteria way easier than plasmid |
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Describe lambda gt-lambdaB and how it gets packaged.
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EcoRI cleavage gives two arms, and foreign DNA inserted to make it 93% (fits in the 75-105% size requirements
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what is cosmid lambda?
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hybrid of lambda phage and a plasmid
accomodates large DNA inserts of 45 kb!! |
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What is a BAC
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Bacterial artificial chromosome
engineered version of ecoli fertility F factor. accomodates inserts of DNA in 100,000-300,000 bp selectable with chloramphenicol resistance put in bacteria by electroporation |
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What is a YAC
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Yeast Artificial Chromosome
accomodates inserts as big as 2,000,000 bp lots of stuff in em that we didn't really cover but that makes them ideal for big genomes |
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What is a genomic digest?
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mechanically sheared fragments of a 3,000,000 kb genome
makes random overlapping fragments which can be PAGEd isolate 15 kb long molecules and add linkers and then insert into lambda phages and then grown in E coli. gives you genomic library |
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Why do we need DNA hybridization?
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Allows us to screen the genomic library
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What is DNA hybridization?
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the recombinant phages are plated on a lawn of bacteria where they like guys grow a plaque.
Nitrocellulose is then applied and we add NaOH to lyse and denature the DNA. 32P probe DNA is introduced and the target sequence is radioaudiographed. |
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How do we know how to make the probe for human hybridization screening?
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take the mRNA from an enriched source of what you are looking for. Make a cDNA from this mRNA and use as probe
use amino acid sequence from target (use sequences with a lot of Trp and Met Synthesize the probes with 32P Use all the probes and isolate and sequence positive clones |
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Why do we need a probe with minimal degeneracy?
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lots of peptides are encoded by several oligonucleotides. Sequences where there is alot of Met or Trp are best because they only have one codon
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Why isn't Ecoli. good for mammalian mRNA?
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E. coli doesn't have the ability to splice, so we have to make cDNA from mRNA (which doesn't have introns)
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What is the difference between SINES and LINES?
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Short intersperced elements (DNA that doesn't code for proteins)
1,000,000 Alu sequences of 300bp Long intersperced elements nearly 1 million LINES up to 10 kbp |
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how do we make cDNAs
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mRNA + oligo T primer with 3'OH + 4 dNTP's
oligo T pairs with poly A at 3' end of mRNA reverse transcriptase makes a DNA complementary to RNA template increase the pH and dissolve mRNA add terminal transferase which adds oligo GGG to 3' end of the ssDNA then add oligo CCCC to make cDNA |
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What antibody do we use in immunochemical screening of cloned cDNA?
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125I antibody
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Describe DNA microinjection method/
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0.1 mm micropipet
yeilds 2% viability foster mother mouse |
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what is GH 21-kd protein?
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rat growth hormone
in rats it is promoted by metallothionein promoter have an activator |
