BCH5413 TEST 2 QUESTIONS AND ANSWERS ALL REVISED AND UPDATED
6 keer bekeken 0 keer verkocht
Vak
BCH5413
Instelling
BCH5413
BCH5413 TEST 2 QUESTIONS AND ANSWERS ALL REVISED AND UPDATED
Methylation: a primitive immune system for bacteria
- hds-R - Answer-- plasmid transforms into cells are protected (restriction enzyme not expressed)
Methylation: a primitive immune system for bacteria
- mcrA/mcrB/mrr - Answer-degr...
BCH5413 TEST 2 QUESTIONS AND
ANSWERS ALL REVISED AND
UPDATED
Methylation: a primitive immune system for bacteria
- hds-R - Answer-- plasmid transforms into cells are protected (restriction enzyme not
expressed)
Methylation: a primitive immune system for bacteria
- mcrA/mcrB/mrr - Answer-degrades foreign DNA that is not properly methylated
- can occur to DNA obtained from mouse/human cells containing CpG methylated DNA
(can interfere with cloning of mammalian genomic DNA)
pCR 2.1 - TOPO - Answer-Tac Pol adds A-overhangs to PCR products
TOPO cloning
- what it stands for - Answer-TOPO = topoisomerase
- contains lacZa (can use blue/white color screening)
- f1 Ori (can make ssDNA)
- amp-r
- pUC Ori
drawback: PCR product doesnt get directionally cloned
PCR cloning: test digest - Answer-- restriction enzyme site in pCR 2.1 TOPO + PCR
product
- 5' -> 3': will yield fragment of 300 and 4600 band
- 3' -> 5': 700 and 4200 band
Protein expression in E. coli - Answer-- clone DNA out of TOPO vector into downstream
plasmid capable of expressing protein
- expression plasmid encodes lacZ gene (IPTG in environment will induce protein
expression); will product beta-galactosidase protein
*can also be done by having a plasmid expression vector, lacZ gene to transformed E.
coli -> protein expressed
Shuttle vector: replication in E. coli and yeast - Answer-- cloning steps can be
performed in E. coli, protein expression takes place in eukaryotic cell (yeast)
,- Ori
- amp-r
- CEN: ensures segregation
- ARS (ori to be copied within yeast)
- URA3 (uracil synthesis; selection)
Yeast shuttle vector: pESC-URA - Answer-URA3 for yeast selection required for growth
on media without uracil
Mammalian shuttle vector
- pcDNA3.1 - Answer-- contains f1 ori (ssDNA)
- amp-r
- pucOri (copying in E. coli)
- pCMV (promoter to express in mammalian cell)
- has epitope tag
- neomycin resistance (selectivity)
*promoter will always be active, does not need to be induced
cDNA library construction
- standard approach steps in isolating a cDNA clone - Answer-1. isolate mRNA
2. convert to ds cDNA
3. choose vector and insert cDNA
4. create phage or bacterial library
5. screen
6. verify identity
Making a cDNA Library - Answer-- isolate mRNA
- make cDNA
- perform second strand synthesis for ds cDNA and add C overhangs (sticky ends)
- clone into vector with complementary G overhangs
- anheal and ligate
Packaging cDNA library into bacteriophage lambda
- problems and solution - Answer-Problem:
- transformation of plasmids (DNA) into bacterial cells is inefficient
Solution
- use bacterial virus (phage) vector, not a plasmid
Packaging cDNA library into bacteriophage lambda
- steps - Answer-- mRNA transcribed into ds cDNA
- ligate ds linker to all ends of cDNA
- can be cleaved with EcoR1 for sticky ends
- complementary overhang vector added
- in vitro packaging to create recombinant virus particles
,- library then used to infect E. coli
*when E. coli is infected with lambda clones you get a lawn of E. coli; where infection
has occured you will see a plaque
Cloning in Charon 4
- useful for? - Answer-- this type of vector accomodates large pieces of DNA
Steps
- after obtaining recombinant DNA combine with lambda packaging system
- infectious phages will infect E. coli and show plaques
Screening a library
- plaque hybridization - Answer-- lay membrane on petri dish and lift off a replica of
plaques
- DNA on filter corresponds to that of the plaques
- block filter with nonspecific DNA or protein and hybridize to labeled probe
- detect by autoradiography
DNA structure - Answer-- nucleic acid bases (purine (A,G) and pyrimidine (C,U,T))
- sugars (ribose, deoxyribose)
- sugars linked by phosphate groups (sugar-phosphate backbone)
*form antiparallel strands
In mammilian DNA, which base has various forms - Answer-cytosine
Chargaff's rule - Answer-A=T and C=G
Is DNA conservative or semi-conservative? - Answer-semi-conservative
- parental sequence and daughter strand bind together
Alternative Double stranded DNA conformation
- A and Z DNA - Answer-A DNA
- dehydrated form
- right handed helix
- 11 bp per helical
- bp tilted 19 degrees
- major groove narrow and deep
- found with dsRNA and RNA-DNA duplex
z-DNA
- >1% of cellular DNA
- favored by G-C repeats and alternating purines-pyrimidines
- left handed helix
- 12 bp per turn
, - bp tilted 9 degrees
- major groove flattened, nearly gone
- bound by poxvirus E3L virulence factor and down regulates apoptosis genes
Non-duplex DNA structures
- cruciform DNA - Answer-- inverted repeat sequence
- favored by excessive negative supercoiling
- AT-rich cruciforms associated with 'fragile' DNA
Alternative modes of base pairing - Answer-Watson Crick:
A--T and C---G
- adenine is at anti-conformation
Hoogsteen:
- adenine or guanine can be flipped to sin-conformation (different H-bonding)
Hoogsteen base pairing
- when can it occur - Answer-- in triplex and quadruplex DNA structures
Triplex DNA - Answer-- pyrimidine rich strand
- negative supercoiling
Chair DNA - Answer-- two G-rich strands
- down regulation of c-myc transcription
Intrinsic Bends in DNA - Answer-- distortion of the ideal B-DNA conformation resulting
from base stacking in the nucleotide sequence
Eg. Adenine-tract DNA results in 20 degree bends
- also can be found in duplex-oligonucleotide model DNA
- causes 23 degree bend and mis-stacking of one GC
- DNA is not a uniform structure
Does B-DNA have a perfect structure? - Answer-No
- sequence specific local variation in twist between paired bases, sugat conformation, tilt
of base pairs, rise distance, etc.
Supercoiled DNA
- negative v. positive - Answer-- left handed under-twisted DNA is in a negative
supercoil
- right handed over-twisted DNA is in a positive supercoil
*most is negatively supercoiled (topoisomerase releaves supercoiling)
Denaturation/Melting of DNA - Answer-- separation of DNA strands
Voordelen van het kopen van samenvattingen bij Stuvia op een rij:
√ Verzekerd van kwaliteit door reviews
Stuvia-klanten hebben meer dan 700.000 samenvattingen beoordeeld. Zo weet je zeker dat je de beste documenten koopt!
Snel en makkelijk kopen
Je betaalt supersnel en eenmalig met iDeal, Bancontact of creditcard voor de samenvatting. Zonder lidmaatschap.
Focus op de essentie
Samenvattingen worden geschreven voor en door anderen. Daarom zijn de samenvattingen altijd betrouwbaar en actueel. Zo kom je snel tot de kern!
Veelgestelde vragen
Wat krijg ik als ik dit document koop?
Je krijgt een PDF, die direct beschikbaar is na je aankoop. Het gekochte document is altijd, overal en oneindig toegankelijk via je profiel.
Tevredenheidsgarantie: hoe werkt dat?
Onze tevredenheidsgarantie zorgt ervoor dat je altijd een studiedocument vindt dat goed bij je past. Je vult een formulier in en onze klantenservice regelt de rest.
Van wie koop ik deze samenvatting?
Stuvia is een marktplaats, je koop dit document dus niet van ons, maar van verkoper Perfectscorer. Stuvia faciliteert de betaling aan de verkoper.
Zit ik meteen vast aan een abonnement?
Nee, je koopt alleen deze samenvatting voor €11,83. Je zit daarna nergens aan vast.
