EXAM 2 Flashcards
two types of bacterial strand
rough and smooth
what experiment hsowed molecular basis of hereditability
the rough vs smooth bacteria
what happens when heat killed smooth is added to rough
rough bacteria transformed into smooth strain
only molecular capable of trasnforming rough bacteria to smooth
DNA
DNA carries what
heritable information
how can you determine whether a comvo of 2 or more molecules owrking together is required for trasnformation
taking one trait out, determines what trait is required for what gene
What Would Happen if Transformation Was Due to Protein or RNA, Rather Than DNA?
the results of the original experiment (before fractionation) would change because the DNA fraction would not have been sufficient for transformation, and treatments like protease or RNase would block the transformation if those molecules were the genetic material
DNA strucutre is composed of a four nucleotide code of what and what do they contain
sugar phosphates with a either a pyrimidine or purine
what are pyrimidines
cytosine or thymine
what are purines
adenine or guanine
DNA strand is polarized with what ends
5’ end and 3’ end
when DNA is double stranded, what happens between the strands
hydrogen bonds form between paired bases
are DNA strands paralllel or anti
antiparalel
5’ and 3’ refers to what
carbon posiiton in the sugar group
5’ end is the 5’ carbon linked to what
phosphate group
3’ end is the 3’ carbon linked to what
OH group
phosphodiester bond links what
5’ and 3’ carbons in the polynucleotide chain
double helix held togehter by what
hydrogen bonds between complimentary base pais
pyrimidine pairs with what
pruine
CG pairs form how many h bonds
3
wich pair forms 2 h bonds
AT
which pair forms 3 h bonds
CG
AT pairs form how many h bonds
2
where do eukaryotes store DNA
in nucleus
DNA and chromatin organized into
chromosomes
Are human somatic cells diploid or haploid
diploid
are human gametes diploid or haploid
haploid
human cells typically have how many of each chromosome
2
how many non sex chromosomes are there
22
what is a nucleosome
DNA wrapped around a core of proteins in the nucleus
nucleosome core is what
histone octamer
what is included in a histone octamer
2 each of H2A, H2B, H3, H4
what model is used to describe nucleosomes
beads on a string
what links nucleosomes together
histone H1
histone H1 compacts nucleosomes into what
chromatin fiber
chromatin fibers fold into what
topological domains
how many sex chromosomes do humans have
2
how many autosomes do humans have
22
how many copies of each autosome are ther
2
how many total pairs of chromosomes are ther
23, 46 total (22x2) +2
what do cells do to chromosomes at begining of mitosis
cells compact their chromosomes and begin to duplicate their DNA
how many chromosomes do human gametes have
23
once cells duplicate their chromosomes, what do they form
sister chromatids
2 copies of the chromosome joined where
centromere
After DNA replication, what happens to cells
metaphase
cells at metaphase have what
sister chromatids
what is the #n for cells at metaphase and expalin what they are
4n, 46 chromosomes replciated to form 46 sister chromatids
how many origins of rep does each chromosome have
many
what form attachemt point for the mitotic spindle
centromeres
centromers form the attachment point for what
mitotic spindle
form special caps at the chromosomes ends
telomeres
during m phase, what link mitotic spindles to centromere and facilitate distribution to daughter cells
kinetochores
when do kinetochores link mitotic spindle to centromere and facilitate distribution to daughter cells
m phase
during m phase, kinetochores link what
mitotic spindle to centromere and facilitate distribution to daughter cells
Fish is used to visualize what and uses what
specific chromosomes or regions of chromosomes within a cell using fluorescently labeled probes
first step of fish
chromosomes isolated and spread onto slide
what is probe design in fish
specific dna probes labeled with a different fluorescent color, designed to bind to unique sequences of the chromosome or chromosomal region of interest
in fish, bound probes are visualized usoing what
fluorescence microscope
You are testing a new method of detecting trisomy 21 in gametes via FISH. What would you design your DNA probe to target?
The probe should target sequences unique to chromosome 21. You can select a region of the chromosome that is present in multiple copies or in a gene-rich area, such as a centromeric region (close to the centromere) or a specific locus that is abundant on chromosome 21.
You are testing a new method of detecting trisomy 21 in gametes via FISH. How would you control for the non-specific binding of your probes?
use a control probe on another chromosome to ensure that the probe is binding specifically to 21
You are testing a new method of detecting trisomy 21 in gametes via FISH. What are the expected FISH results if a metaphase cell you are testing is positive or negative for trisomy 21?
if negative for trisomy 21, there will b e2 signals, one for each chromosome… is positive there will be 3 signals indiciating an extra chromosome
probes laveled by synthesizing them with what
nucletoides conjugated to fluorophores
fluorophores do what
absorb photons at a shorter wavelength (higher Energy) to reach excitation adn then emit them at a longer wavelength (lower
photon absorption causes what
electron to trasniton to a higher orbit and enter an excited state
what happens to the electron when returning to the ground state
returns to lower orbit and emits a photon at longer wl
what is the longer wavelength due to in fluorescence emission
energy being lost from the vibration of molecules aka heat
what is the stokes shift
phenomenon of excitation resulting in longer wl emission
in nucleotide additon, what basepairs with its partner in th etemplate strand
nucleoside triphosphate
the incoming nucleoside triphosphate does what
base pairs with its partner in the template strand
what is incoming nucleoside triphos covalently attached to
free 3’ hydroxyl end of growing DNA strand
New DNA is syn in what direction by what
5’ to 3’ via DNA polymerase
energy for the polymerization comes form what
hydrolysis of high energy phosphate bond in incoming nucleoside triphos and release of pyrophosphate
how are incorrect nucleotides recognized
imporper base pairing
what happens when DNA poly runs into improper bp
DNA poly stops and cleaves it from the strand, then replaces with correct nucleotide
chromosomes are replicated at what
multiple replication origins
each origin is made of what
2 replication forks
each rep fork ahs what kind of strands
leading and lagging strand
lagging strand must be made initially as a series of what
short DNA strands called okazaki frag
what happens to strands when they reach opposite form
they are switched
DNA replication is primed by what
RNA primers
DNA poly requires what as a starting point
based paired 3’ end
Does DNA primase requrie a base paired 3’ end
no
DNA primase uses what to make RNA primers in the 5’ to 3’ direction
ribonucleosides
at end of chromosome, there is no room for what
new okazaki fragment for lagging strand
what if the problem of not having room for new okazaki frag was not resolved
chromosomes would lose the sequences at their ends wit heach replication getting shorter and shoerter
telomerase does what
uses RNA template to extend DNA synthesis
what repairs nicked DNA
DNA ligase
what exactly doesDNA ligase do
hydrolyze ATP to attach AMP to the 5’ phos of the nick
what is used to join okazaki frag
DNA ligase
common DNA lesions
depurination, depyrimidination, cytosine deamination
what is deamination
changing a cytosine to a uracil
conseqeunces of deamination
adeneine will bind uracil during replicaiton, results in bp substitution of a G to an A
what is depurination, when does it occur
pruine bases removed from the DNA molecule, occurs when glycosidic bond between purine base and sugar is broken, leaving apruinic site
consequences of depurination
when rep machinary finds missing purine, it can skip to the next complete nucleotide, results in one daigher DNA double helix missng one nucleotide pair
what causes pyrimidine dimers
UV radiation
consequences of pyrimidine dimer fomration
stalls DNA poly during replicaiton, can lead to mutations, DNa breaks, or cell death depending on number of dimers
for base excision repair, what removed deaminated cytosine
uracil DNA glycosylase
in base excision, uracil DNA glycosylase removes what
deaminated cytosine
in base excision repar, sugar phosphate backbone with missing base is cut by what
AP endonuclease, phosphodiesterase
in abse excision repair, what is the single nucleotide gap filled and repaired by
filled by DNA poly, repaired by DNA ligase
for nuc excision repair, what are 2 mechanisms
bacterial and human
for bacterial mech of nuc excision, what happens,
multienzyme complex recognizes DNA lesion, one cut is made on each side of the strand leaving a 12 nt gap, helicase removes the strand, DNA poly and ligase repair the gap
differences in human mech of nuc excis repair
once damage recognized, helicase is recruited to unwind the DNA duplex, then excsion occurs leaving a 30 nt gap
restriction enzymes cleave what at what
DNA at specific sequences
restriction enzymes often work as what
dimers
for restriction enzymes, the cut position is often what around the central point
symmetrical
for restriction enzyems the cut posiiton is often what around the central point
symmetrical
the sequence for restriction enzymes to bind to is often what
palindromic
what is palindromic
read the same on each antiparallel strand
2 ends that restriciton enzymes can produce
blunt ends and staggered ends
staggered cuts make what
sticky ends
what are special about sticky ends
they can join back toghether
palindrome is applied to regions of DNA with what
inverted repeats
sequence when the inverted repeat occurs within each individual strand
mirror repeat
what is a mirror repeat
seqeunce when the inverted repeat occurs within each individual strand
when inserting DNA frag into plasmid, they are both cut with what
restriction enzymes
when inserting DNA frag into plasmid, what are added to seal nicks in the backbone once sticky ends repair
DNA ligase and ATP
when inserting DNA frag into palsmid, DNA ligase and ATP do what
seal the nicks in the backboen once the sticky ends repair
ligation of reagments with staggered overhands does what and uses what
joining two DNDA frag with sticky ends using DNA ligase and ATP
fragment with staggered overhangs is joining two fragments cut by what
same restrction nuclease
conversion of staggered to blunt end for ligation is joining tow fragments cut by what
difference restriction nuclease
recombinant DNA plasmids can be introduced into bacteria via what
transformation
what is recombinant DNA
artifically created DNA the combines genetic material from different sources or species
when amplifiying clone region in bacteria, what happens
bacteria is culutred and replicates plasmid millions of times as the bacteria replicates its own DNA
Howcanweensurethatonly bacteria that contain our plasmid of interest are replicated?
use selective markers
what can be used to generate a genomic library
recombinant DNA
large fragments can be digested with a restriciton enzyme whose site…
occurs at a sifficient frequency in the genome
what are used to synthesize specific DNA regions
Primers
what is step one of first cycle of amplification
heat to separate strands
step 2 first cycle of amplification
cool to anneal primers
step 3 of first cycle fo amplification, what is added also
DNA synthesis, DNA poly, dATP, dGTP, dCTP, dTTP
what method amplifies DNA regions exponentially
PCR
how many DNA molecules produced with second cycle of amplificatin
4 double stranded DNA molecules
in gel electrophoresis, what will happen to more repeats
they move less
CODIS Profiling via STR fingerprinting what is it
uses Short tandem repeats to create unique DNA fingerprint for identifying individuals
what is amplified in STR fingerprinting
STR loci via PCR
in STR fingerpringing, how are DNA fragments sperated
by size using gel electrophoresis
each indiivuadl has how many alleles per STR locus
2
what is codis
national forensic database
how is STRs used
if crime scene DNA sample has same STR pattern, it matches the suspect
true or false: rasing the annealing temp in a pcr reaction decreases the off target binding of primers and inc specificity
true
generation of labeled DNA probes produces what
mix of hexanucleotides
what are hexanucleotides
set of 6 random nuc annealed togehter
how are probes made visbale
take nucleotide and add
what is a DNA probe and what is it used for
short single stranded DNA sequence labeled with detectable marker, used to hybridize to a complementary DNA sequence
how to generate labeled DNA probe
choose target DNA sequence, amplify it via PCR or cloning, label the probe, purify the labeled probe, verify the probe
how are hexanucleotides related to DNA probe labeling
hexanucleotides used to bind randomly across template, dna poly extends them, generatees a labeled probe that covers entire seqeunce
DNA sequencing uses to do what
figures out nucletoide seqeunce
what is added in first step of DNA seqeuncing
primer
what is different about ddntp
missing OH on 3’- prevents linkage of next AA
difference between dntp and ddntp
ddntp is chain terminating, has missing OH at 3’, dntp can allow extension at the 3’ end
are primers used in DNA sequencing random
no
which is greater in conc, dNTP or ddNTP
dNTP, to allow chain growth
Primers are custom - synthesized in vitro. What happens if you include additional sequence in the 3’ end of the primer that is not present in the target region you want to amplify?
there will be no amplification bc the 3’ end wouldnt be complimentary to the target
add additionlal seqeunce that isnt present in target region we want to amplify… What happens if you instead add this sequence to the 5’ end of your primer?
the 5’ to 3’ wont be affected, but when the reverse trasncritpase comes through to replicate that initial strand, the additional seqeucne will now be incorporated into our target
nucleotide consists of what
sugar phosphate+base
nucleoside consists of what
sugar phosphate
nucleotide subunits held together by what
phosphodiester bonds
hydroxyl group is what end
3’
phosphate group is what end
5’
couling of 2 strands around each other creates what
2 grooves, wider groove called major groove, narrow groove called minor groove
organisms differ due to what
different nucleotide sequence
complete store of info inan organism DNA
genome
human genomes contain about how many protein encoding genes
20K
in euk, dna enclsoed where
in nucleus
nucleus formed by what
2 concentric lipid bilayer membranes
outer nuclear membrane is connected to what
ER
nuclear envelope supported internally by what
network of int filaments called nuclear lamina
what are nuclear lamina
thin feltlike mesh just beneath inner nuclear membrane
nuclear DNA divided up into what
chromosomes
what is chromatin
compelx of DNA and tightly bound protein
how do bacteria normally carry genes
carry them on a single DNA molecule thats usually circular
each human cell besides gametes contains what
2 copies of each chromosome, 1 from mom 1 from dad
maternal and paternal chromosomes of a pair are called what
homologous chromosomes
what are the only non homologous chromosomes
sex chromosomes in males (XY) (Y inherited from dad, x from mom)
each human cell contains how many chromosomes
46
what do the 46 chromosomes in human cell consist of
2 x 22 common to both male and female, 2 sex chromosomes
what isa karotype
display of all 46 human chromosomes
similar species genomes can vary greatly due to what
selection pressures
what percent of human genome codes for proteins
1%
difference between intron and exons
introns are removed during RNA splicing, exons stay in the final mRNA and determine protein structure
each DNA molecule that forms linear chromosome must contain what
a centromere, 2 telomeres, replication origin
what happens during interphase
genes are expressed, chromosome replicated, sister chromatids produced
what are mitotic chromosomes
highly condensed chromosomes in a dividing cell
once DNA rep is complete, what does the cell do
completes m phase, then mitosis occurs
what happens during m phase
chromosomes condense, nuclear envelope breaks down, mitotic spindle forms
what is the replication origin
location where duplication of DNA begns
what is centromere
allows 1 copy of each duplicated and condensed chromosome to be pulled into each daughter cell when a cell divides
what is a kinetochore
forms @ centromere, attaches duplicated chromosomes to mitotic spindle in manner that causes sister chromatids to be pulled apart
what are telomeres
ends of chromosomes, continued repeated nuc seqeunces that allow ends of chromosomes to be fully replicated
keeps polymerase firmly on DNA when its moving but releases it as soon as it runs into double stranded DNA
sliding clamp
what is clamp loader
open and close the ring in a regulated manner
what does clamp loader hydrolyze
ATP as it loads new clamp onto primer
what protein opens the helix
DNA helicase
proteins at replicaiton fork cooperate to form what
replication machien
how many polymerase work on leading strand
1
how many polyermase work on lagging strand
2
what are used to begin each okazaki frag and what makes them
short RNA primers, DNA primase makes them
close association of all proteins does what for replication
increases efficiency
DNA replication machine does what to unsealed Okazaki frag and why
leaves them behind , they still contain RNA that primed their synthesis at the 5’ end
what happens to the left behind okazaki frag
the RNA primer is removed, gap filled by repair enzymes
difference between euk and bacteria DNA rep
euk uses 3 diff types of DNA poly
which strand uses poly E
leading strand
which polymerase are used on lagging strand
poly A and poly S
which polymerase used on leading strand
poly E
poly E binds to what sepcificaly
both the sliding clamp and replicative helicase
poly a includes what
DNA primase
poly S does what
completes synthesis of each Okazaki fragment
how many poly SU in bacteria
1
strand directed mismatch repair system does what
removes replication errors that remain in the wake of the replicatoin machine
mutator genes do what
increase rate of spontaneous mutation
strand dir mm repair picks out mismatches from normal DNA how
monitoring potential to distort the doubel helix
consequences of dna poly incorporating ribonucleotide instead of deoxy and not repairing it
causes weak links, high mutation rates, genome rearrangement
which conc is higher, ribonuc, deoxyribo
ribonucleotides
prevent DNA tangling during replication
topisomerase
what actually is topisomerase and what does it do
reversible nuclease that adds itself covalentyl to DNA backbone phosphate, breaks phosphodiester bond
what does topoisomerase I do
produce single strand break, allows 2 sets of DNA helix on either side of nick to rotate freely using phospho bond in opposite the nick as a swivle
what does topisomerase II do
forms covalent linkage to both strands of DNAhelix at the same time, making doubel strand break
what activates topisomerase II
sites onchromosomes where 2 double helices cross over each other
DNA synthesis begins where
replication origins
what are replication origins
positions at which helix is first opened
what DNA is morelikely to be found at rep origins
AT
how many oriigns of rep do bacteria have
1 origin
what can reveal the pattern of Labeled DNA
autoradiography
how many origins used each time human cell divides
30K to 50K
what phase does DNA repl take place
S PHASE
euk origins are licensed for rep by what
assembly of an origin recog complex
origin sequence contains binding site for what
ORC, rich in AT
origins located in proximity to each other tend to do what
fire together
what ensures that each region of DNA is replicated only once
properties in the ORC
what prevents the binding of helicase
phosphoryalting ORC
new nucleosomes assembled where
behind replication fork
histones mainly syn in what phase
S phase
why are histones mainly syn in S phase
level of histone mRNA increases about 50x
is histone mRNA stable
no
what monitors level of free histone ensuring amount of histone made=amount of new DNA synthesized
tight linkage between DNA dyn and histone SYN
what require histone chaperones
orderly and rapid additon of new h3-h4 tetramers and h2a-h2b dimers behind fork
termination of DNA rep occurs via what
ordered disaseembly of replication fork
what causes main termination signal in euk
head on encounter with a fork moving in opposite direction
what causes disassembly/ removal from DNA when 2 forks meet
helicase @ each fork covalentyl modified by addition of ubiquitin
why cant final RNA prumer synthesized on lagging strand template be repalced by DNA
there is no prumer ahead of it to provide a 3’ OH end for repair poly
what is seqeunce of repeat in telomeres in human
GGGTTA
what does telomerase do
replenishes these repeats each time a cell divides
what does telomerase use as a template
RNA template that is a componenet of itself
telomeres packaged into specialized structures that do what
protect ends of chromosomes
when chromosomes break in half what happens
theyre repaired quickly
telomeres must be distinguised from what and why
chromosome breaks because they would be attemtped to be repeaired, fusing chromosomes
in telomeres, what chews back and what is expsoed
specialized nuclease chews back @ 5’ end of telomere leaving protruding single strand 3’ end
protruding end of telomere attracts what adn whats its name
group of protein tha form protective chromosome cap called sheltertin
protects telomeres from being treated as damaged DNA
sheltertin
telomere length regulated by what
cells and organism
in most human somatic cells what happens to telomres over time
they shorten
DNA repair fixes things caused by what
heat, metabolic accidents, radiation, exposure to enviromental substances
inc rate of what when repair gene inactivated
mutation
human diseases linked w diminished capacity for what
DNA repair
xtreme sensitivity to UV radiation
xeroderma pigmentation
what is DNA purination
DNA of each human cell loses about 18K purine bases bc n glycosul links to deoxyribose break
depurination can remove what
guanine or adenine
what is deamination
cysteine to uracil causes thymidine dimers
if there was no DNA repair, what would happen
damage/mutations would be passed down
why is double helix suited for repaur
carries 2 seperate copes of all geneti info, one on each strand
2 most common methods of DNA repar
base excision, nucletodie excision
base excision starts with what
DNA glycosylase remove incorrect nucletoide sugar phos
what cuts out the incorrect nuc sugar phos in base excison
AP endonuclease and phosphodiesterase
gap made in base excision filled by what
DNA polymerase and ligase
in bacteria, what is nucleotide excision
one cut made on each side of damage, associated helicase removes entire portion of damaged strand
expalin process of nucleotide excison in humans
damaged DNA is recog, helicase locally unwinds DNA, excisoin nuclease enters and cleaves on either side of the damage; leaves gap of about 30 nucletodies to be filled in
coupling nucleotide excisoin repair to transcription ensures what
cell’s most important DNA is efficientyl repaired
what facilitates damage detection
chemistry of DNA bases
Explain DNA repair in an emergency
highly accurate DNA poly stall when they get to the damage, emergency cells deploy versatile but less accurate polyermases
what are the versatile but less accurate backup polys called
translesion polymerase
risks that translesion poly pose
wrong base substitiution, single nuc deltion, probbaly generates mutations
what is an especially damgerous DNA damage type
both strands of double helix are broken leaving no template to be used for accurate repair
what can cause double strand breaks
ionizing radiation, replication errors, oxidizing agents, metabolites
what would happen if fouble strand breaks werent repaired
breakdown of chromosomes and loss of genes when cell diviided
2 mechanisms evolved to deal with double strand breaks
non homologous end joining, homologous recombination
simple process of non homologous end joinign
broken ends processed to remove any damaged nucleotides, brought together and rejoined by DNA ligation
what results at site of breakage in nonhomolog end joining
usually a mutation
what can non homolog end joining occasionally create
rearrangement in which one broken crhomosome becomes covalently attached to each other
predominant double strand fix in humans
non homologous end joinign
orderly progession of cell cycle stopped when and starts back when
damage detected, restarts only when damges has been repaiared
in mammels, damage can block entry to which phases
G1 to S
what is ATM protein
large protein kinase that generates intracellular signals needed to halt cell cycle in response to many type of NDA damage
what can defects in ATM protein cause
unrepaired DNA lesions
how is homologius recombo used in DNA damage/repair
helps repair double-strand breaks (DSBs) in DNA by using a homologous sequence as a template
what guides homologous recombo
DNA bp
homologous recombo takes place only between what
DNA duplexes that have extensive similariites
before udnergoing homolog recombo, what happens
two DNA helices will sample each others DNA seqeunce by testing potential bp betwee single strand from one DNA duplex and a comp single from another
when is homologous recombo initiated
when a match is found between the 2 helices (doesnt need to be perfect)
how accurate is homologous recombo
VERY, no loss or alteration of nuc at site of repair
in homolog recombo, what carry out strand dance
broken DNA and template duplex
what is strand dsance
one of the damaged strands can use comp strand as template once theyre close, ends of broken DNA cleaved back
when is homolog recombo more likely to occur and why
during S and G2, specialized nuclease for it becomes extremely active
initiation nuclease of homolog recombo in euk
MreII
what does MRE II do in homolog recombo
chews back in 5’ to 3’ direction, leaves protruding 3’ ends
in homolog recombo, what protects exposed end and form what
single strand bp coats single strans protect from other nucleases in cell
strand exhange directed by what
RecA/Rad 51 proein
what is RecA used in
ecoli
what isi Rad51 used in
eukaryotes
what loads Rad51/reca onto protruding DNA single strand
group of accessory proteins
what is DNA held bu in strand exhange
Rad51/ RecA
how is DNA split apart in strand exhange
complex grasps nearby DNA duplex, stretches it, destabilizes it and pulsl it apart
what kind of protein is RecA/Rad51
ATPase
RecA/Rad51 requires what
each monomer along filament has to be ATP bound
homologous recombo can rescue what
broken and stalled forks
why would fork be stalled
fork attempts to move through certain types of DNA damage, clogs up rep machinary
repair by homolog chromosomes introduce what risk
repairs can use wrong portion of genome as template
explain loss of heterozygosity
repair using homolog from other parent instead of sister chromatid- can convert the sequence of repaired DNA from materal to paternal or vice versa
homolog recombo used to do what, in order to do what
used to exhange material between 2 different chromosomes in order to generate DNA molecules that carry novel combos of genes
meiotic recombo begins how
with double strand break
in meiotic recombo, what breaks both strands of the DNA in one of the recombing chromosomes-> stays attached
SpoII
what does MREII do
chews back on DNA ends, removing proteins along with DNA and laving protruding 3’ single strand ends needed for strand inversion
what are holiday juncitons
four-stranded DNA structures that form as an intermediate during homologous recombination
what is branch migration
expands region of heteroduplex that was init created by strand exhange reaction
what is crossover control
ensures roughly even distribution of crossover points along chromosomes -> ensures each chromosome undergoes at least 1 crossover event every meosis
alternative versions of the same genes
allleles
what is gene conversion
divergence from their expected distribution during meiosis
ability to manipulate DNA with percision in a test tube or organism known as..
recombinant DNA technology
what do restriction nucleases do
cut large DNA molecules into speciifc fragments
what defines where restruction nucleases cut
specific sites defined by the local nucleotide sequence
restriction nucleases can vary based on
species of bacteria they came from
why are restriction enzymes useful in lab
each enzyme will always cut a particular DNA molecule at same sites
what are the two staggered cutting restriction nucleases
ecoRI and HindIII
seperates DNA molecules of different sizes
gel electrophoesis
each nuc carreis hwat charge
negative
to seperate DNA molecules larger than 500 nucleotide pairs, what is used
gel made of agarose
to seperate DNA molecules less than 500 nuc pairs, what is used
polyacrylamid gels
what is special about pulsed field gel electrophoresis
direction of electric field changes periodically
DNA bonds on agarose or polyacrylamide gels are invisable unless…
DNA is labeled or stained in some way
purified DNA molecules can be labeled how
with radioisotopes, chemical markers in vitro
what is DNA clongin
act of making many identical copies of a DNA molecule, amplification of a DNA seqeunce
simplist way to clone DNA
insert frag of DNA into purified DNA of self replicating genetic elements
in cloning, what is the first thing that happens to the purifid plasmid
cut with restriction nuclease
how is the cloned DNA recovered
after cells are lysed, plasmid isolated, it is cut out of plasmid DNA with same restriction nuclease that was used to insert it
what is a cDNA clone
copies made by reverse trasnscriptase of retroviruses
what is advantage of cDNA clone over genomic clone
they contain the uniteruptted coding sequence of a gene
success of cloning in PCR relies on what
selectivity of DNA hybridization and ability of DNA poly to copy a DNA template faithfuly through repeated rounds of replicaiton
what is now the method of choice for cloning short DNA seq
PCR
how sensitive is PCR cloning
can detect a single DNA molecule in a sample of at least part of that sequence
what purpose do oligonucleotides do in cloning
used as probes and primers for PCR
