Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
204 Cards in this Set
- Front
- Back
|
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 |
plasmids
modif bact viruses |
|
|
dna
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 |
t
|
|
|
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?
|
retroviruses
|
|
|
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
|
sticky
|
|
|
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
|
|
|
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?
|
BAC
|
|
|
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
|
|
|
cloning
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 |
|
|
cloning
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 ? |
reinfection
|
|
|
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?
|
introns
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 ?
|
prey
|
|
|
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 |
lysed
|
|
|
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
|
|
|
probe
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
|
|
|
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? |
RNA
|
|
|
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
|
dna
|
|
|
northern blotting is when ____________ is transferred to nitrocellulose
|
rna
|
|
|
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? |
RFLP
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
|
steps
adjacent |
|
|
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
|
|
|
ddNTP
|
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
|
phosphodiester
|
|
|
dideoxy seq
you will prepare four different ? |
cocktails
|
|
|
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 |
up
|
|
|
dideoxy seq
a labeled _____ is used to initiate DNA synthesis |
primer
|
|
|
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
|
|
|
ORF
|
open reading frame
|
|
|
pcr is used to
|
amplify the amount of dna that you have
|
|
|
pcr
you use primers at the ___________ of the target g on the 2 strands |
3'
|
|
|
pcr
______________ flank the targeted sequence |
the primers
|
|
|
pcr
_________ makes new dna |
taq polymerase
|
|
|
pcr
the first 2 strands will be ____________ |
variable length, bec they dont have a common stop signal
|
|
|
pcr
when a primer attaches to its target site on the seq it? |
anneals
|
|
|
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
|
|
|
alkaptonuria
urine turns? |
black when expos to sun
|
|
|
alkaptonuria
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 6. |
|
|
scientists first found the g for alkaptonuria in?
|
aspergillus
|
|
|
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
|
|
|
alkaptonuria
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? |
amniocentesis
|
|
|
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? |
MsII
|
|
|
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
example? |
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 |
|
|
transgene
|
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 5 |
|
|
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
|
g
|
|
|
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 |
t
|
|
|
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
|
|
|
yeast
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
causes? |
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
|
grow
|
|
|
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
|
mouse
|
|
|
c elegans is a?
|
nematode
|
|
|
c elegans has gonads that contain _________ c?
|
syncitial
|
|
|
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
|
