BCH5413 TEST 2 QUESTIONS AND ANSWERS ALL REVISED AND UPDATED
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BCH5413
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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
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