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56 Cards in this Set

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How does EcoR1 cut?
What kind of a sequence is this?
G^AATTC
Palindrome
What is DNA stained with during gel electrophoresis? What kind of fragments travel faster?
EtBr, smaller
What enzyme joins DNA fragments together?
DNA ligase
What was Muller's experiement?
- 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
What is the Ames Test?
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
What does restrictin enzymes leave?
3' OH and 5' P
ECORI
endonuclease, cuts 5bp palindromic

G^AATTC
PSTI
6 pb seq

5' CTGCA^G 3'
3' G^ACGTC 5'
SMAL
6 bp seq

5' CCC^GGG 3'
3' GGG^CCC 5'

leaves blunt ends
How does Ecoli avoid cutting its own DNA?
It methylates nucleotide bases and it only cuts un-methylated DNA
How is DNA structutre?
Antiparallel double helix
Draw skeleton of DNA, note the difference between RNA and DNA and DDNTP
2' OH - ribose
2' H - deoxyribose
3'H - dideoxyribose
How many rings do Purines have? What na are purines? Draw basic structure
2 rings
AG
6 member - 5 member
How many rings do Pyrimidines have? What na are they? Draw basic structure
1 ring
CT
6 member
Describe the Griffith "transformation" experiment
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!!
Describe the Avery experiment
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
Describe the Hershey-Chase "blending" experiment
bacteriaphage radioactive labeling with sulfure (protein) and phosphorous (DNA) showed that infection DNA enters cell but protein does not
How many H bonds are between the 4 Nucleiacids?
AT - 2 H BONDS
CG - 3 H-BONDS
What are the major and minor grooves of DNA? What strand is the Watson strand?
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'
What was the Meselson and Stahl experiment?
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
What are the 3 stages of DNA replication?
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
Name 6 enzymes of DNA replication and their function.
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)
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
Delbruck - Luria experiement
Mutations occur at random and are not due to stress
What are 3 ways that mutations occur?
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"
How does proofreading work?
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
What is complementation and a complementing group?
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
What are balancer chromosomes?
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
Benzer/T4 experiment
found genes are in a linear arrangment

rII+ -- grow
rII -- not grow

overlapping muations never get wildtrype
What is unique about TATAA.. sequences?
30% of cells have this signal, associated with TFIID (transcription factor IID), a collection of proteins
What are trans-acting factors and enhancers?
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
What are the 3 stages of DNA transcription?
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
What are found in Eukaryotic cells that are unqiue to DNA transcription?
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
What is the purpose of splicing?
alternative splicing, in humans 3-5 alternative splice forms/ gene, can generate diversity
Southern Blotting's purpose
to find how many inserts are in a certain colony


#lines = # of inserts
What are the 4 parts of the pUC19 plasmid?
1. origin of replication
2. antibiotic resistence gene
3. Lac Z
4. unlike bacteria, plasmids can be maintained in multiple copies per cell
What 4 possibilties can arise from mixing enzyme cut plasmid and DNA?
1. 2 pieces of DNA stick together
2. DNA forms a circle
3. pUC19 seals back up
4. pUC19 + DNA
What does Lac Z and Xgal do?
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
What should be added in a Sanger's solution?
1. DNA polymerase
2. Template strand
3. Free ATCG
4. RNA primer (matches Lac Z)
5. DDNTPS
What does degernate mean in terms of translation?
multi codons correspond to an amino acids

4^3 > 20 aas
What did Crick + Brenner discover?
Frame shift muations --> 3 frame shifts = 1 new aa so codons are 3 nucleotides/amino acid
What is the starting codon?
What is the terminating codon?
starting - AUG (METHIOINE)
stop - UGA, UAG, UAA
What is UTR?
URT = untranslated region, independent of protein translation, only for transcription!
What is TRNA?
TRANSFER RNA - attached to ribosome

3' - AA
anticodon 3'-5'
reads 5'-3'

tRNA - sythentases - joins aa to tRNA
How big is the human genome?
3 billion bps
What is used to stain DNA during gel electrophoresis
EtBr, aromatics and hydrophobic so sticks to double helix, it is also a mutagen
What is important about PCR?
Need 2 different primers on opp. strans! Amplication of targeted sequence
What are the procedures for PCR?
1. start with a pool of DNA + 2 primers
2. denature (heat)
3. Anneal primers
4. extend primers
5. repeat
6. specific amplication
What enzyme is used in PCR?
TAQ - polymerase from a thermophilic bacteria, can withstand high temps needed to denature DNA
How many replications does it take to get DNA fragment of choice?
2
What are 5 different Cloning vectors?
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
What is so special about ddNTPS?
Lack a 3' OH and cannot be extended by DNA polymerase
What kind of a gel is used for Sanger sequencing?
polyacrylamide instead of agarose
What is the name of the graph that shows the output of all four colors ?
Electropherogram,
What is shotgun sequencing?
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)
What is the key idea with shotgun sequencing?
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