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genetics chapter 11 gene isolation


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genomics is
extension of dna tech to global analysis of nucleic acids
live cells is

in vitro
in vivo
in vivo
in vitro means/
in a test tube
in vivo
donor dna is put inot
modif bact viruses
in vivo
vectors do what?
acces chrom like plasmids that carry and amplify the gene of interest
donor dna is cut up using?
restriction endonucleases
restr endonucl cuts the ___________ of the dna
sugar-phosphate backbone
recombinant dna consists of?
frag of donor dna and cut vector chromosomess like plasmids
dna cloning is when?
the recomb dna molec are copied every time the bact divides and grows
in vitro
gene of interest is found by?
binding of spec short primers to the ends of the desired seq
dna tech depends on what 2 basic foundat of molec bio research?
1. ability of spec prot to rec and bind spec dna seq
2. abilit of ss dna or rna to unite to form double strands
what are 3 types of donor dna?
1. genomic dna
2. cDNA
3. chemically synthez DNA
genomic dna is?
straight from live organ
it will need to be cut up bef cloning
what do you have to do to genomic dna bef using it?
cut it up
cDNA is?
ds version of a mRNA molec.
does not have introns
t or f
cDNA does not have introns
why do researchers want to use cDNA and not just mRNA?
mRNA is unstable and its hard to amplify mRNA
cDNA is made from mRNA using?
reverse transcriptase
reverse transcriptase originally came from?
what are the steps in making a ds cDNA from mRNA
1. put a short olig(dT) chain on the poly A tail of mRNA
2. reverse transciptase makes complementary DNA strand. it has a hairpin loop
3. degrade original mRNA strand w NaOH
4. DNA polymerase uses hairpin loop as primer and copies the DNA
5. cleave loop w S1 nuclease
synth of ds CDNA from mRNA

the CDNA strand made by reverse transc has a ?
hairpin loop
synth of ds CDNA from mRNA

the hairpin loop on cDNA strand acts as a primer for?
DNA polymerase I
synth of ds CDNA from mRNA
S1 nuclease does what/
cleaves hairpin loop on cDNA strand
synth of ds CDNA from mRNA

NaOH is used for?
degrading origianl mRNA strand
why doesnt NaOH degrade both the mRNA and DNA?
i dont know, gots to ask the prof
chemic synth DNA

why would you do this?
if you cant isolate the desir g by other means
cDNA was used to make insulin bec?
bact cant remove introns.
a restriction enzyme will cut dna into?
restriction fragments
restriction enzmes make __________ ends
EcoRI is from
e coli
dna palindrome means?
both strands have the same seq but in antiparallel orientat
ECoRI makes cuts only betw the ?
G and A nucleotides on each strand of palindrome
which restrict enzyme cuts between G and A
ECORi makes cuts that are sticky ends what does that mean?
that they are single strands that can "stick" to a complementary seq
SmaI make s flush cuts and leaves?
blunt ends
SmaI , which leaves blunt ends can be used for recombinants if you also use?
another special enzyme to join the blunt ends togeth
you would want to digest the donor and vector dna with the same?
restriction enzyme. so they will have matching ends
after donor and vector dna have hybridized what else needs to be done?
close the sugar phosphate backbone using DNA ligase
DNA ligase does what/
closes the sugar phosph back of dna
in order for the insulin g to be transcribed in a bact it has to be inserted next to?
bacterial regulatory regions
amplificat inside bact c

set of amplified copies of single donor DNA fragm is called/
recombinant DNA clone
what factors do you need to consider for cloning vectors
1. small
2. replicate a lot in bact
3. have good restrict enz sites
4. have a way to identify and recover the recombin molec
what can you use as vectors?
1. plasmids
2. bacteriophage vectors
describe a bacteriophage vector?
can hold up to 50 kb DNA
plasmid designed as vector for dna cloning

insertion into pBR322 is detected by?
inactivation of one drug resist gene (tetS)
TetS means that?
it has been inactivated by insertion of DNA fragment
what can you use for dna inserts larger than 25 to 30 kb?
1. cosmids
2. PAC
3. BAC
4. YAC
cosmid is?
hybrids of lambda phage DNA and beact plasmid DNA. this is inserted into lambda phage particles.
in c form circluar molec that replicate extrachrom
cosmid is a hybrid of?
lambda phage dna and bacter plasmid dna
cosmids are inserted into?
lambda phage particles
PAC is
P1 artificial chromosome.
PAC works like a ?
cosmid but can accept inserts from 80 to 100 kb
PAC is a derivat of ?
bacteriophage P1.
compare bacteriophage P1 to lambda ?
bacter P1 has a larger genome than lambda
BAC is?
bacterial artificial chromosome
BAC are derived from?
F plasmid
BAC can carry inserts in size?
150 to 300 kb
which vector is the "workhorse " for large scale cloning?
YAC is
yeast artificial chromosomes
YAC can fit dna of what size?
larger than 300 kb
plasmid expression vector does what?
has bact promoters that init transcr at high levels when a allosteric regula is added
which vector has special bact promoters that init transcr at high levels when an allosteric regulator is added?
plasmid expression vector
dna gets into bact by what 2 ways?
1. transformation
2. transduction w phages similar to cosmids. they then circularize.
3. transduct w phages that then infect and lyse the c
how do you transform?
put bact in solut that has the recomb DNA molecule
entry of recomb molec into bact c

when lambda phage is used, you will have repeated rounds of ?
recovery of recomb molec

in bact how do you do this?
sep from larger bact chrom by centrifuge
genomic library is made by
take all the dna from a genome, cut it up w restr enzy and insert each segment into a vector
what kind of libraries can you have?
1. cDNA library
2. genomic library
CDNA library represents?
only the protein coding regions of genome
how do you make a comprehensive cDNA library?
get mRNA samples from
1. diff tiss
2. diff devel stages
3. organisms grown in diff enviro condit
A cDNA library will lack?
untranscribed regulat seq
finding a spec clone

what are the 2 types of probes?
1. recog spec nucleic seq
2. those that recog a spec protein
probes can be thought of as "bait" for the much larger ?
identificat of spec clone

you will transfer the colonies or plaques to ?
an absorbent membrane
identificat of spec clone

colonies or plaques on the nitrocellulose are _____ and the DNA is denatured
identificat of spec clone

after lysing the colonies, you bathe them w?
solut of ss probe spec for DNA being sought
identificat of spec clone

the position of a positive clone will be made obvious by?
position of concentr readiactive or flourescent label
autoradiogram is?
an exposed piece of x ray film that will have a dark spot showing where probe was concentrated
where does DNA for a probe come from?
1. cDNA from tiss that expresses the the g
2. homolog g in related organ
3. prot product of g of interest
4. labeled free RNA
clone by phone is when?
you get a homolog seq from another organism that your colleague has.
Protein product--- back translate to dna codon---- ?
then make a synthetic DNA
if you make probe from a protein product what do you have to watch out for?
degeneracy of dna code. you choose a short stretch of aa with minimal degeneracy
you use a "cocktail" of oligonucleotides when you derive your dna probe from?
the prot pred of the g of interest

labeled free rna can be used only when?
you have a nearly pure populat of indentical molec of rna. like rRNA
probes for finding proteins

you need what 2 things?
1. express library
2. antibody t spec prot prod of the g of interest
probes for finding prot

how do you make an express library?
by putting cDNA int o vector in correct triplet reading frame with a bact protein.
probes for finding prot

expresson vector will be translated to make a?
fusion protein translated reg of cDNA and a part of normal beta- galactosidase
probes for finding prot

after making fusion proteins what will be used to detect the correct protein?
an antibody to that protein.. and another LABELED antibod will connect to the first Ig.
probing to find a spec nucleic acid in a mixt

what is most common method?
blotting starts by?
1. sep molec by gel electrophoresis
gel electrophor
dna frag will migrate at a speed ?
inversely dependent on their size
how do you visualize bands on gel electrophoresis?
using ethidium bromide
how do you figure out the absolute size of each restrict frag in a mixture?
by comparing its migrat dist with a set of standard frag of known sizes
southern blotting

to do this you have to first?
denature the DNA and use a membrane to blot the gel after electrophor
southern blotting

you hybridize the membrane w?
a labeled probe
northern blotting

can be used to detect?
whats one applicat of northern blotting?
to see if a spec g is transcribed in a cert tiss
all of these tech uses the ability of nucleic acids to?
bind comlementary nucleotide sequences
southern blotting is when ________ is transferred to nitrocellulose
northern blotting is when ____________ is transferred to nitrocellulose
functional complementation or mutant rescue

depends on having _________ in the g of interest
recessiv mutation
functional complementation or mutant rescue

you detect the correct clones from the bact library by?
seeing which clone will restore the funct of the recessive mutation
functional complementation or mutant rescue

first step will be to?
make a bact or phage library with wild type a+ recom donor DNA inserts
functional complementation or mutant rescue

you transform c of ________ by using DNA from indiv clones in the libary
recessive mutant c line a-
positional cloning is?
any meth of finding a spec clone by figuring out where it is on the chromosome
what 2 things are needed for positional cloning?
1. genetic landmarks to set boundaries on where g might be
2. ability to investig the contin segm of dna extend betw the delimiting genetic landmarks
positional cloning

what can serve as landmarks?
or other molecular polymorphisms, chromosomal break points
positional cloning

___________ can be used to find and order clones falling betw genetic landmarks
chromosome walk
positional cloning

chromosome walk is?
you keep using overlapping probes to move away from an identified landamark
positional cloning

you use the ________ of a new set of clones as probes for finding another set of clones further away from original marker and towards the target
end fragments
chromosome walking is a series of _____ from one ________ to the next
to do efficient chrom walking you need to know?
how the array of clones that hybridize to a given probe overlap each other
how do you figure out how an array of clones overlap each other?
by using a restriction map
in the book,
they used ________ to generate a restriction map
2 diff enzymes and then compared the different pieces to see which one had to be in the middle.
you can use ______ to determine the base seq of a dna segme
dideoxy sequencing
dideoxy triphosphate
a ddNTP does what what to DNA synthesis?
it blocks it
a ddNTP lacks?
the 3' hydroxyl group
a ddNTP prev the ____________ bond from forming
dideoxy seq
you will prepare four different ?
dideoxy seq

final steps of process are?
1. display fragments in size order using gel electrophor
2. label the strands. label either the primer or the indiv ddNTP
dideoxy seq
you read __________ the gel
dideoxy seq

a labeled _____ is used to initiate DNA synthesis
if you use a differ flourescent color emitter for each of the ddNTP then you can use?
automated DNA seq machines
in a printout from an automatic seq

N represents a?
base that cannot be assigned
in a printout from an automatic seq

each of the peaks would correspond to what on a gel?
a dark band on the gel
after seq a piece of DNA how can you tell how many g it has?
have a comput scan for initiation and stop codons
a stretch of g identified as having a start and stop codon is called a ?
open reading frame
open reading frame
pcr is used to
amplify the amount of dna that you have
you use primers at the ___________ of the target g on the 2 strands
______________ flank the targeted sequence
the primers
_________ makes new dna
taq polymerase

the first 2 strands will be ____________
variable length, bec they dont have a common stop signal
when a primer attaches to its target site on the seq it?
taq polymerase comes from what organism?
thermus aquaticus. lives in thermal vent
pcr can amplify dna that is in ___________
very small amounts
disadvantages of pcr

to design the primers?
you have to have a little bit of info about the seq
disadvant of pcr

fragments have to be?
smaller than 2kb

urine turns?
black when expos to sun

what enzyme is absent?
homogentisate 1,2 dioxygenase
HGO stands for?
homogentisate 1,2 dioxygenase
aku what steps did they do to learn about it?
1. examined symptoms
2. did mendalian analysis of its inher patterns
3. mapped the g
4. isolated it from aspergillus
5. used g from aspergillus to find homolog seq in human chrom
6. the cDNA finds the g in lambda genomic library
7. do pcr of exons from g

scientists first found the g for alkaptonuria in?
jose canon looked thr ___________ for a match to the HGO g of the aspergillus
human cDNA
scientist used the clone they ident from the cDNA to ?
hybridize it to the human chrom. it hybridized to band 3q2

scientist used cDNA close to ?
get the full length g from a genomic library
scientists tested a family that had alkaptonuria by?
using pcr to amplify the exons of their HGO g
detect human dis alleles

for humans we need to able to identify ____________?
heterozygous prospect parents
detect human dis alleles

taking fetal c and culturing them is used in?
detect human dis alleles

name 3 common disease that can be ident by amniocent?
1. cystic fibrosis
2. duchenne musc dystrophy
3. tay sachs
detect human dis alleles

chorionic villus sampling is?
you take a small sample of c from the placenta. it can be done earlier than amnio
detect human dis alleles

you can identify some dis by looking at ______________ differences
restriction site
the __________ or ___________ of a restriction site can tell you if you have the dis g
presence or absence
restriction site difference

give an example?
sickle c anemia

mutat eliminates a a cut site for rest enzyme MsII
restrict site differe

the mutat in sickle c anemia can be recog by cutting w restrict enzyme?
the mutat in sickle c anemia, after being cut by MsII will show up on ?
southern blotting
since most dis causing mutat dont cause ch in restr enzyme sites, what else can you use/
probe hybridization
diag muat by probe hybridiz

synth probes can be made that can detect even?
a differ in a single base pair
diag muat by probe hybridiz

alpha1-antitrypsin deficiency. increases probab of devel pulmonary emphysema. has only a single base ch
diag muat by probe hybridiz

a wild type seq probe ______________ to the mutant seq of alpha1-antitrypsin when applied at high temp on a southern blot
will not bind
detect human dis alleles

what tech can you use?
1. amniocent
2. chorionic villus sampling
3. restr site differe
4. probe hybridization
5. pcr tests
genetic engineering is?
use of recomb dna tech to alter an organ genotype and phenotype
give 2 exampl of genet engin?
1. goats that secr antib derived from fungus in their milk
2. plants that have a fish "antifreezing" g
is the g that is transf in genetic engineering
transgenic organism
organism that has had the transgene put into it.
how can you put a transg into an organ?
1. transform
2. inject
3. bact or viral infect
4. hit it w dna coated tungsten or gold part
a gen inserts ectopically.
this means that?
it means that it inserted at a site diff than the original resident g
what is the best well stud euk genetic model?
s. cerevisiae
YIps are?
simple yeast vect deriv from bact plasmids
YIps can be used for ____________ replacement
how can you detect a g replac in a yeast?
plating c on a medium that selects for a marker allele on the plasmid
t or f
bacterial plamsids will not replicate in yeast on their own
a bact plasmid must __________ to replicate in a yeast
integrate into yeast chromosome
yeast have a natural yeast plasmid called?
2 micron plasmid
YEp is a?
yeast plasmid with the replic origin from the 2 micron plasmid
YEp stands for?
yeast episomal plasmid
A YEp can have both a __________ and ____________ replication origin
bacterial and yeast
like bact, some yeast have ?
a plasmid, called 2 micron
to make sure the spindle apparatus of the yeast segregates your vector plasmid you can add?
yeast centromer
YCp is?
yeast centromere plasmid
YAC is made up of?
yeast centromere, replicative origins, linear dna (not circular), has yeast dna telomeres
a YAC is

a. linear
b. circular
a. linear
a 2 micron plasmid is

a. linear
b. circular
b. circular
yeast vectors can be what 3 things?
1. integrative
2. autonomously replicate
3. resemble artif chromos
GMO stands for?
genetically modified organisms
what vector is used to prod transgen plants?
Ti plasmid
Ti plamsid comes from?
agrobacterium tumefaciens.
agrobacterium tumefaciens

crown gall disease
agrobacterium tumefaciens
causes dis in plants by?
inserting into plant dna and causing growth of tumors
T-DNA from Ti plasmid makes what when inserted into plant chrom?
nopaline. something the bact needs for growth
whats a a problem with using Ti plasmids?
they are too large to be used easily. you have to make it in steps.
Ti plasmid

a bact intermed vector with transgene + TDNA will make?
a cointegrate plasmid
Ti plasmid

cointegrate plasmid will use kanamycin resistance as?
a selectable marker
plants with genet engineered TDNA will ____________ on a medium containing kanamycin
plants . TDNA

how can you detect presence of the insert?
1. screen the pl tiss for transgen gen markers
2. pres of nopalin
3. screeing purif DNA with a TDNA probe in a south hybrid
gene engin in animals

what are 3 common animals?
1. c elegans
2. drosophilia
3. mus musculus- mouse
mus musculus is a
c elegans is a?
c elegans has gonads that contain _________ c?
c elegans

syncitial c have?
many nuclei
e elegans
the transgenic dna is injected into?
the syncitial region of one of the arms of the gonads
c elegans
inject transg dna will form?
extrachromosomal arrays that are indep units outside the chrom
c elegans

extrachromosomal arrays are?
indep units outs the chrom

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