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56 Cards in this Set
- Front
- Back
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How does EcoR1 cut?
What kind of a sequence is this? |
G^AATTC
Palindrome |
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What is DNA stained with during gel electrophoresis? What kind of fragments travel faster?
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EtBr, smaller
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What enzyme joins DNA fragments together?
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DNA ligase
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What was Muller's experiement?
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- Xrays cause mutations, longer dose = more lethal mutations, DNA is like a physical object and can be affected the same way that other molecules are affected
fruit flies |
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What is the Ames Test?
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His- (can't make his) and His + colonies
1. based on invertants 2. want to make sure bacteria isn't repairing on its own --> repair- phenotype 3. permeable walls to make easier contact with DNA + chemicals 4. mix in liver enzymes to recreate stomach environment - figure if something causes mutations or not Salmonella |
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What does restrictin enzymes leave?
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3' OH and 5' P
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ECORI
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endonuclease, cuts 5bp palindromic
G^AATTC |
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PSTI
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6 pb seq
5' CTGCA^G 3' 3' G^ACGTC 5' |
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SMAL
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6 bp seq
5' CCC^GGG 3' 3' GGG^CCC 5' leaves blunt ends |
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How does Ecoli avoid cutting its own DNA?
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It methylates nucleotide bases and it only cuts un-methylated DNA
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How is DNA structutre?
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Antiparallel double helix
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Draw skeleton of DNA, note the difference between RNA and DNA and DDNTP
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2' OH - ribose
2' H - deoxyribose 3'H - dideoxyribose |
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How many rings do Purines have? What na are purines? Draw basic structure
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2 rings
AG 6 member - 5 member |
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How many rings do Pyrimidines have? What na are they? Draw basic structure
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1 ring
CT 6 member |
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Describe the Griffith "transformation" experiment
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streptococcus pneumonia
Mutant colonies lacks sugar coat & makes rough colonie that can be visually distinguishable, also lack of coat allows immmune system to clear R strain --> Mice survive Wild type "s" - due to capsule bacterial colonies are smooth and kills mice R --> dies S--> Lives Heat-killed S --> lives Heath S + R --> Dies transformation is stable, inherited state, does not need cell to transfer DNA!! |
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Describe the Avery experiment
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1. take heat-killed S cells and separate into proteins, RNA, DNA
2. test diff fractions for ability to transform 3. Only enzymes known to degrade DNA had an effect FOUND ONLY DNA was the material |
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Describe the Hershey-Chase "blending" experiment
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bacteriaphage radioactive labeling with sulfure (protein) and phosphorous (DNA) showed that infection DNA enters cell but protein does not
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How many H bonds are between the 4 Nucleiacids?
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AT - 2 H BONDS
CG - 3 H-BONDS |
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What are the major and minor grooves of DNA? What strand is the Watson strand?
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Major --> protein fitst nicely, most DNA interaction
Minor --> protein can't fit rotation by ~36 produces helix (1/10) of turn Watson - top 5'-3' Crick - bottom 3'-5' |
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What was the Meselson and Stahl experiment?
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semi-conservative (AB, AB)
conservative (AA, BB) disperisve (mix of AB) heavy (AA), hybrid (AB), light (BB) after many gen 1. lots of light, lil hybride 2. no hybrid 3. always hybrid |
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What are the 3 stages of DNA replication?
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iniation - unwinding DNA + exposing both strands to become templetes
Elogation - incorporation of nt into newly synthesized complemntary strand Termination - release of duplicate double-helices replication is bidirectional |
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Name 6 enzymes of DNA replication and their function.
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1. Helicase - unwind + open DNA, dydrolyze ATP for energy
2. DNA polymerase - acts only on single stranded DNA extending 5'-3' 3. RNA primase - makes primers for lagging strand 4. DNA ligand - join pieces, removes RNA primer, cut, fixes and glues 5. Topoisomerase - unwinds DNA 6. Telomerase - makes sure chromosomes replicates always to the very end (stem cells, tumorous cells, germ cells, active telomerase) |
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Substitution
Transition Transversion Insertion + Deletions Inversion Translocation |
Substiution - way one nucleotide for another
GAATTC --> GACTTC Transition - C -> T, A -> G, switch to other ring memeber faster and easier Transversion - pyr - pur Insertiona + Delections - covalent break/rejoin Inversion - flip ABC top --> CBA bottom Translocation - biggest scale, break/join chromosomes |
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Delbruck - Luria experiement
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Mutations occur at random and are not due to stress
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What are 3 ways that mutations occur?
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1.Depurination - bases can be lose but polymerease can replace complimentary na, but during replication strans are open and polymerase don't know what to put there
2. Deamination - C --> U, change one pyrimidine to another 3. UV light - makes one double base, a thyimine dimer, gets cut and polyermiase repairs "excision repair" |
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How does proofreading work?
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notices engery differences
G - T (the G is less favorable due to energy stability) exonuclease - chew back the DNA strand and tries again Markings for paretnal strand so it knows waht to fix, enzymes come along and add markers, so it needs to fix right after snythessis of na |
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What is complementation and a complementing group?
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two mu complement if they are on different genes, they do not complement if they are on the same gene
a complementation group is a collection of mus that DONOT complement |
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What are balancer chromosomes?
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m/m --> LEATHAL
M+/M -> M+/M+ OR M/M+, never produce mutatant offspring, helps to maintina M in population never recombines because recombinant offsprings die |
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Benzer/T4 experiment
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found genes are in a linear arrangment
rII+ -- grow rII -- not grow overlapping muations never get wildtrype |
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What is unique about TATAA.. sequences?
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30% of cells have this signal, associated with TFIID (transcription factor IID), a collection of proteins
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What are trans-acting factors and enhancers?
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trans-acting factors are encoded somewhere else in genome from the starting signal
enhanchers are cis-regulatory module, cis to the gene, controls trans acting factor |
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What are the 3 stages of DNA transcription?
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1. iniation - find beginning of gene, open DNA to make it 1 strand
2. elongation phase - RNA polymerase II is catalyzing new adition of a new nucleotide then shifting over by 1 3. Termination - signals to stop |
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What are found in Eukaryotic cells that are unqiue to DNA transcription?
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1. addn of 5' cap
2. addn of poly-A-tail, poly-A-polyermerase adds As to 3' end 3. splicing done by spicesome (recongize donor and acceptor and cut in middle) spliceosome - proteins and RNA exons - keep introns- cut |
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What is the purpose of splicing?
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alternative splicing, in humans 3-5 alternative splice forms/ gene, can generate diversity
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Southern Blotting's purpose
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to find how many inserts are in a certain colony
#lines = # of inserts |
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What are the 4 parts of the pUC19 plasmid?
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1. origin of replication
2. antibiotic resistence gene 3. Lac Z 4. unlike bacteria, plasmids can be maintained in multiple copies per cell |
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What 4 possibilties can arise from mixing enzyme cut plasmid and DNA?
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1. 2 pieces of DNA stick together
2. DNA forms a circle 3. pUC19 seals back up 4. pUC19 + DNA |
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What does Lac Z and Xgal do?
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Lac Z encodes a beta galactodsidase enzyme. In the presence of Xgal, this enzyme can change it to blue. If LacZ is interrupted by insert, then the cells grow to be white
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What should be added in a Sanger's solution?
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1. DNA polymerase
2. Template strand 3. Free ATCG 4. RNA primer (matches Lac Z) 5. DDNTPS |
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What does degernate mean in terms of translation?
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multi codons correspond to an amino acids
4^3 > 20 aas |
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What did Crick + Brenner discover?
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Frame shift muations --> 3 frame shifts = 1 new aa so codons are 3 nucleotides/amino acid
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What is the starting codon?
What is the terminating codon? |
starting - AUG (METHIOINE)
stop - UGA, UAG, UAA |
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What is UTR?
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URT = untranslated region, independent of protein translation, only for transcription!
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What is TRNA?
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TRANSFER RNA - attached to ribosome
3' - AA anticodon 3'-5' reads 5'-3' tRNA - sythentases - joins aa to tRNA |
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How big is the human genome?
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3 billion bps
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What is used to stain DNA during gel electrophoresis
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EtBr, aromatics and hydrophobic so sticks to double helix, it is also a mutagen
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What is important about PCR?
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Need 2 different primers on opp. strans! Amplication of targeted sequence
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What are the procedures for PCR?
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1. start with a pool of DNA + 2 primers
2. denature (heat) 3. Anneal primers 4. extend primers 5. repeat 6. specific amplication |
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What enzyme is used in PCR?
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TAQ - polymerase from a thermophilic bacteria, can withstand high temps needed to denature DNA
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How many replications does it take to get DNA fragment of choice?
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2
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What are 5 different Cloning vectors?
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1. Plasmids: <10kb, Ok for mRNAS
2. Lambda phage: 15-20 kb, easy to make 3. Cosmids, Fosmids: 40 kb inserts 4. Bacterial artificial chromosome (BACS): 20-200 kb 5. Yeast artificial chromosomes (YACs): can hold megabases; useful for eukaryotic seq |
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What is so special about ddNTPS?
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Lack a 3' OH and cannot be extended by DNA polymerase
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What kind of a gel is used for Sanger sequencing?
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polyacrylamide instead of agarose
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What is the name of the graph that shows the output of all four colors ?
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Electropherogram,
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What is shotgun sequencing?
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1. purify DNA from genomic region of interest
2. Shear or cut (partial digest) the DNA 3. Sequence the ends of many random inserts (can use sam primer) 4. Sequence enough fragments to cover the region of interest 5. since fragments are redundant, they will o verlap works well for modest size regions (BAC) |
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What is the key idea with shotgun sequencing?
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Use paired end sequencing. Use two primers to sequence both ends of an insert. These two 500-1000 bp sequences fragments are then a. on opposite strands and b. a known distance apart
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