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131 Cards in this Set

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thcut DNA at specific recognition sequences, producing fragments joined with DNA vectors to form recombinant DNA molecules
restriction enymes
replicate autonomously in host cells & facilitate the manipulation of newly created recominant DNA molecules
vectors
vectors are constructed from
often bacterial plasmids and phages
host cells commonly used for replication
bacteria, yeast, mammalian cells
method for amplifying a specific DNA sequence present in a collection of DNA sequences (e.g. genomic DNA)
polymerase chain reaction
the PCR method allows DNA to be cloned without
host cells
DNA sequence analysis methods
restriction mapping, DNA sequencing, Southern blotting
uses hybridization to identify genes and flanking regulatory regions within cloned sequences
Southern blotting
In 1971, Danna and Nathans described the
isolation of an enzyme from a bacterial strain & use of the enzyme to cleave viral DNA at specific nucleotides sequences
recombinant DNA
formed by joining two heterologous molecules - usually produced by in vitro ligation of DNA from two different organisms
restriction enzymes (endonucleases)
generate specific DNA fragments in a sequence - cleaves or nicks DNA at those sites, derived from a variety of microorganism
human genome contains
more than 3 billion nucleotides and 25 000 to 30 000 genes
restriction fragments' size depends on
how often a given restriction enzyme cuts the DNA -
if all 4 nucleotides are present in equal proportions enzymes with a four base recognition sequence will cut how many times?
on avg. every 256 base pairs (4^n - 4^4=256) - this is many small fragments
NotI hase an ___ base recognition sequence while AluI has a ____ sequence.
NotI
why do actual fragment sizes produced by cuting DNA with a restriction enzyme vary?
number and location of recognition sequences are not always distributed randomly in DNA
palindromes in DNA typical of
recognition sequences can be read from either strand when read in the 5' to 3' direction
recombinant vectors
vectors carrying an inserted fragment
one of the first restriction enzymes to be identified
from E. coli strain R - EcoRO w/six-nucleotide recognition sequence
DNA fragments produced by EcoRI digestion have
overhanging single-stranded tails "sticky ends" that can form hydrogen bonds with complementary single-stranded tails - once bonded can be covalently linked to form recombinant DNA molecules
plasmid vectors were
the first vectors developed; from naturally occuring extrachromosmal double-stranded DNA molecules that replicate autonomously w'in bacteria
plasmid vectors are modified to
increase their copy number so can get several hundred copies & engineered to contain convenient restriction enzyme recognition sequences & marker genes so can identify them in cells
pUC18 is? advantages?
a plasmid vector; small so it can carry large DNA inserts, has an origin of replication & produces up to 500 copies of fragments per cell, has polylinker in a lacZ gene
polylinker
engineered region of vector containing large number of restriction enzymes
how does the polylinker of pUC18 aid in identification
in a lacZ gene - disrupts the lacZ gene resulting in white colonies (normal expression give blue colonies) when grown on Xgal
lambda phage
genetically modified strains are used as vectors to carry pieces of DNA larger than 10kb - ~ 20kb. Genome has een completely sequenced and mapped - middle 3rd can be replaced w'out affecting ability to infect cells & form plaques
how do you clone DNA using a lambda vector?
purify and cut phage DNA using restriction enzyme (e.g. EcoRI) - cut into 3 chromosomal fragments: L&R arms and disposable middle
arms isolated & mixed with foreign DNA cut with same enzyme

DNA ligase produces recombinant vectors

vectors packaged into phage protein heads in vitro & added to bacterial hosts

after replication creates plaques by rupturing host
what is a plaque
clear area on otherwise opaque bacterial lawn caused by growth & reproduction of phages from which cloned DNA can be recovered
cosmid vector
hybrid vectors from combining parts of lambda chromosome with parts of plasmids - contain the cos sequence of phage lambda, pplasmid-derived antibiotic resistance gene
what is the cos sequence of phage lambda
necessary for packaging DNA into phage protein coats and for replication
cosmids replicate as
plasmids
cosmids can carry DNA inserts much larger than lambda vectors (50 kb vs. 10-15) because
most of the lambda genome has been removed
shuttle vectors
e.g. animal viruses like SV40 - hybrid vectors - contain genetic markes that are selectable in boh types of host cells and used to shuttle DNA inserts between E. coli & another type of host cell (e.g. yeast)
bacterial artificial chromosome (BAC)
a vector with a large cloning capacity based on the fertility plasmid (F factor) of bacteria used to act as vectors for eukaryotic DNA & can carry 300 kb inserts
F factors are capable of
capable of independently replicating plasmids that transfer genetic information during bacterial conjugation - carry fragments p to 1 Mb in length
BAC vectors contain
F factors genes for replication & copy number

at least 1 antibiotic resistance marker

polylinker containing clustered recognition sequences

polylink flanked by promoter sequences used to generate RNA molecules for the expression of the cloned gene for use as probes in chromosome walking & sequencing the insert
expression vectors
engineered to produce many copies of a selected protein in a host cell - avail. for both pro & eukaryotes e.g. pET for use in E coli host
use of pET expression vector
gene to be copied clined into a recognition sequence in the polylinker next to T7 viral promotor and lac operon

host cell genome modified to carry T7 vral polymerase, lac promotor and he lac operator

expression induce by adding lactose analog IPTG to medium, displacing repressor from lac operator, activating T7 polymerase gyrase gene on the bacterial chromosome AND the gene in polylinker

T7 polymerase binds to the T7 promotor & transcribes the gene in the polylinker
IPTG
analog of lactose placed in medium to displace repressor from lac operator leading to transcriptioon of inserted protein-coding fragments in pET
T7 polymerase
activated by IPTG activity on the lac operon - binds to T7 promotor & transcribes gene in polylinker of expression vector IPTG
4 steps required to create recombinant DNA molecules & transfer to E coli host
1. isolation of DNA to be copied, treatment w/ enzyme to make fragments

2. fragments ligated to plasmids cut with same enzyme - recombinant vector

3. vector transferre to E. coli when replicates

4. bacteria lated on nutrient medium, form colonies, screened for presense of recombinant plasmids
K12
laboratory strain of E. Coli that is genetically well characterized & can host wide range of vectors - cells in colonies derived from single ancestral cell so all cells in coliny & teir plasmids are genetically identical
phages containing foreign DNA are used to infect host E coli - when plated, each resulting plaque represents _____ .
a cloned descendant of a single ancestral bacteriophage
Saccharomyces cerevisae
yeast widely used as host for cloning & expression of eukaryotic genes
5 reasons to use yeast as a eukaryotic host
1. can be grown much like bacteria
2. genetics widely studied - large catalog of mutations good gene map
3. entire genome has been sequenced & most genes ientified
4. can posttranslationally modify proein to it folds into fxn'l form to study fxn - bacteria can't
5.safe organism for producing protin for vaccines & therapeutic agents
examples of recombinant proteins synthesized in yeast
Hep B virus surface proein
malaria parasite protein
epidermal growth factor
platelet-derived growth factor
alpha1-antitrypsin
clotting factor XIIIA
yeast artificial chromosome (YAC)
a yeast cloning vector - has telomeres at each end, an origin of replication & a centromere joined to slectable marker genes TRP1 & URA3 + a cluster of respriction enzyme recognition sequences
yeast chromosomes can carry ______ & so are useful to genome projects
100-1000 kb in YACs
transformation
DNA transfer when vector is a plasmid
transfection
DNA transfer using a viral vector
agrobacterium tumifaciens
soil bacterium - infects plant cells and produces tumors (plant galls) in many plants - source of Ti plasmid - used to transfer recombinant
plant cells carrying a recombinant Ti plasmid can be grown in tissue culture to form
a cell mass called a callus that can be induced to form roots & shoots & eventually a mature plant carrying a foreign gene
T-DNA
section of the Ti plasmid (tumor-inducing) from A. Tumifaciens that control tumor formation & compounds required for growth of infecting bacteria - foreign genes can be inserted here & placed in plants by infection with A. tumifaciens
transgenic organisms
contain foreign gene(s)
methods for transferring DNA into mammalian cells
endoytosis, YACs, infection be retrovirus vectors - usually DNA then integrated into host genome
advantage of YACs as vectors for mammalian cells
increases efficiency of gene transfer into mice for research - transfer YAC into nucleus of fertilized egg or stem cell - makes transgenic mice
dsDNA
double-stranded DNA
how do retroviral vectors function?
single-stranded RNA transcribed by reverse transcriptase into a dsDNA molecule that integrates into host genome & is passed to daughter cells - virus can be engineered to remove viral genes so can accept forieng DNA including human
one application of retrovirus-based vectors
gene therapy
polymerase chain reaction
developed by Karl Mullis - rapid method of DNA cloning that eliminates need for host cells
how PCR copies specific DNA sequences
use sequence of desired gene to synthesize two oligonucleotide primers (one for 5', one for 3' end) - primers bind to complementary nucleotides flanking tge sequence to be cloned - heat-stable polymerase added after hybridization to synthesize second strand of DNA; repeat
3 steps of PCR
1. denaturation
2.primer annealing
3.extension by polymerase
primer annealing
temperature lowered to between 50-70 deg. C (annealing temperature)so primers bind to single strands flanking DNA to be copied
denaturation of DNA -
heat 90-95 C - DNA dissociates into single strands in about 5 min
Taq polymerase
heat-stable form of DNA polymerase added to primed single-stranded DNA - DNA synthesis carried out 70-75 C - extends the primers
extension (in PCR)
Taq polymerase extends primers by adding nucleotides in the 5' to 3' direction, making double-stranded copy of target DNA
advantages of PCR over cell-based cloning
rapid - takes a few hours, not days

design of primers can be done with computer

commercial synthesis of oligonucleotides is fast & cheap

sensitive & amplifies from tiny DNa smaples - can use samples that are partially degraded or embedded in a medium e.g. amber
uses for PCR
genetic testing, forensics, moleclar paleontology

screening for mutations in genome, identifying markers, can synthesize allele-specific probes for genetic testing
limitations of PCR
info about nucleotide sequence of target must be known
minor contamination of sample with DNA from other sources causes problems e.g. shed skin cells from cop at crime scene
genomic library
contains at least one copy of all the sequences in a genome, constructed using host cell cloning sine PCR fragments are small
how big does a genomic library have to be to have 95-99% chance og containing all sequences in a genome?
N=ln(1-P) / ln(1-f)

where

N is number of required clones
P probability of recovering a given sequence
f is fraction of genome in each clone
flow cytometry
technique used to prepare cloned libraries from individual human chromosomes
how does flow cytometry work?
chromosomes isolated by staining metaphse chromosomes with 2 fluorescent dyes for AT and GC pairs - flow past a laser beam that causes them to flouresce & photometer sorts & fractios by dye binding & light scattering
pulsed-firled gel electrophoresis
version of gel electrophoresis used to isolate chromosomes for chromosome-specific libraries
unexpected discovery from sequencing chromosome III of yeast
~50% of genes on this chromosome were unknown - showed mutagenesis and traditionally gene mapping very inefficient
cDNA library
contains DNA copies make from mRNA molecules present in a cell population at a given time, representing genes transcriptionally active at the time - isolates poly-A 3' tail bearing mRNA on
why is cDNA so named
complementary to the mRNA active in cell population at the time
how are clones in a cDNA library different from genomic library
introns are removed, mRNa doesn't include sequences near gene that regulate it
steps in producing cDNA
mixing mRNAs with poly-A tails with oligo-dT primers to form a partially double-stranded product that is extended by reverse transcriptase to form a complementary DNA copy - mRNA-DNA double-stranded hybrid acted on by RNAase H to make primers used by DNA plymerase I that makes DNA & removes RNA primers
RNAase H
partially digests RNA leaving fragments that are primers for DNA polymerase I
how to clone cDNA
into plasmid or phage by attaching linker sequences to ends that are recognized by restriction enzyme to make sticky ends
linker sequences?
short double-stranded oligonucleotides containing restrction enzyme recognition sequence
reverse transcriptase PCR (RT-PCR)
reverse transcriptase generates single-stranded DNA copies of mRNA, product put in PCR to copy it into double-stranded molecules, then to amplify these copies - Taq polymerase & random primers added to single-stranded cDNA
why use RT-PCR?
more sensitive than conventional cDNA preparation & can identify mRNAs present in only one or two copies of cell
probe
labeled DNA or RNA sequence complementary to some part of a cloned sequence in the library - used to screen a library to recover clones of a specific gene
why can clones carrying human ribosomal genes be recovered from a library using frog Xenopus laevis DNA probes?
ribosomal gene sequences are highly conserved
how to screen a plasmid library
clones grown on nutrient agar plates; replica of the colonies made by pressing a nylon filter onto plate's surface - filter's bacteria then lysed, denatured & resulting single-stranded DNA bound to the folter to be screened with probe - double-stranded hybrid molecules will form for complementary strands & can be assayed PR if used radioactive probe can lay X-ray film on filter that will expose spots where useful colonies are
plaque hybridization
used to screen phage library

solution with recombinant phages spread over bacteria to form plaques that are transferred to a nylon membrane to bedenatured & probed
phage plaques are more efficient for use in screening large genomic libraries than plasmid colonies because
plaques are much smaller so more can be screened on a single filter
restriction map
establishes the name, order & distance between restriction enzyme cleavage sites along a cloned segment of DNA - can serve as identity tags
restriction map units
bp - base pairs
kb - kilobase pairs
importance of restriction maps
can define boundaries of a gene, dissect internal organization of a gene & locate mutations

used to map genes to specific human chromosomes & regions of chromosomes
if restriction enzyme recognition sequences is closely linked to a mutant allele that can be used as a marker in genetic carriers to
identify carriers of recessively inherited disorders or prenatially diagnose a fetal genotype
Southern blot method
detects hybrids; used to identify which clones in a library contain a given DNA sequence & find sizes of chromosomes, or determine whether a clone contains all or only part of a gene
2 components of southern blot technique
separation of DNA fragments by gel electrophoresis & hybridization of fragments using labeled probes
to make a southern blot DNA in the electrophoresis gel is denatured with ___ to ___.
alkaline treatments to form single-stranded fragments
to make a southern blot, transfer of DNA fragments to a membrane is achieved by
placing the membrane & gel on a wick (sponge)in buffer sol'n - layers of blotting paper placed on he filter & weighted - capillary action of buffer through the gel
in the southern blot, how is the filter prepared for hybridization
placed in heat-sealed bag with a labeled single-stranded DNA probe for hybridization; excess probe washed away & hybridized fragments visualized on film
northern blot
like Southern blot but RNA is boud to a filter
western blot
procedure involving proteins bound to a filter
use of northern blot
info about expression of specific genes used to study paterns of gene expression in embryonic tissues, cancer & genetic disorders, detection of alternatively spliced mRNAs, measuring size of mRNA transcripts by comparison with a known marker RNA
James Sanger & colleagues developed
the most commonly used method of DNA sequencing that converts DNA to single strands used for synthesizing a series of complementary strands, each of which randomly terminates at a different, specific nucleotide. This produces a series of fragments that can be separated by electrophoresis & analyzed to figure out the sequence
glyphosate
herbicide effective at very [low] nontoxic for humans & rapidly degraded in soil - resistance transferred to crops like soybeans & maize - inhibits chloroplast enzyme EPSP synthase
EPSP synthase
enzyme inportant in amino acid synthesis - w'out it plants die - EPSP gene cloned from glyphosate-resistant E coli placed in plant leaves to grow resistant calluses formed by A tumifaciens
three major GE crops in USA
maize, soybeans & cotton
golden rice
nutritionally-enhanced rice - extra beta-carotene with 2 genes from daffodil & one from bacteria for use in Asia & Africa where vitamin A deficiency is rampant
environmental risks of GE crops
cross breeding with wild plants, invasiveness of the modified plant, loss of biodiversity
ethics of GE crops
possible allergies, cost for farmers as many GE crops don't produce viable seeds, labelling issues & who regulating bodies should be
insulin production in bacteria
original method of insulin production ; synthetic genes for A & B subjunits constructed by oligonucleotide syntesis & inserted adjacent to the lacZ gene (for beta-galactosidase) - produces fusion polypeptide with amino acid sequence for galactosidase attached to sequence coding for insulin subunit; these purified from bacterial extracts treated with cyanogen bromide that cleaves the fusion protein from the beat-galactosidase - the two subunits spontaneously unite when mixed to make an active insulin molecule
conditions treatable by recombinant pharmaceuticals
heart failure, hypertension
hemophilia
cancer hepatitis
diabetes
disadvantage to using prokaryotic hosts to synthesize eukaryotic proteins
bacterial cells can't correctly process & modify many eukaryotic proteins; can't add sugars & phosphate groups needed for full activity

eukaryotic proteins produced in prokaryotic cells often don't fold properly
how to overcome difficulties involved in synthesizing eukaryotic proteins?
second-generation methods
second-generation methods
use eukaryotic hosts - instead of grwoing in culture get proteins from milk of livestock
second-generation methods in treatment of emphysema
deficiency of alpha1-antitrypsin associated with the heritable form - gene coding for this protein cloned next to a sheep promoter sequence activating transcription in milk-producing cells - fusion gene injected into sheep zygotes in vitro, zygotes placed in surrogate dams - milk produced contains high []
Dolly was loned to facilitate
establishment of a flock of sheep that consistently produces high levels of human protein
Pompe disease
progressive & fatal autosomal recessive condition; metabolic disorder; children have poor muscle tone, oft. die before 2 from respiratory or cardiac complications
how can Pompe disease be treated with recombinant tech
treated with recombinant alpha-glucosidase produced in rabbit milk, now in clinical tests
subunit vaccine
consists of one of more surface proteins of the virus or bacterium - antigen stimulates production of antibodies against teh virus/bacterium

e.g. hepatitis B vaccine
2 types of commonly used vaccines
inactivated (from dead samples) & attentuated (live that can't reproduce)
production of the hepatitis B vaccine
gene for hep B surface protein clined into YEC & produced in yeast host cellsm extracted & purified
pro & con of subunit vaccine
+:source of pure vaccine manufactured in controlled env't

-: developing countries have problems manufacturing, transporting 7 storing; most need refridgeration & injection under sterile conditions
prenatal diagnosis of sickle-cell anemia
single amino acid substitution in beta-globin chain eliminates a cutting site for restriction enzymes MstII and CvnI & so changes pattern of fragments seen on Southern blts (large band vs. 2 smaller)- can also use to identify parental genotypes & genotypes of other family members who may carry gene - use fetal cells from CVS or amniocentesis; family members can be teted with blood sample
MstII
cuts 3 times in the region of normal beta-globin gene - to small fragments - in sickle cell the middle MstII site is eliminated so get one large fragment
If a fetus has a large band and two small bands what is their genotype for sickle cell?
Heterozygous carrier
what percentage of all point mutations can be characterized by restriction enzyme analysis?
5-10 percent
what is necessary for synthetic oligonucleotides to be used as probes to detect mutant alleles?
well-characterized mutant gene; mutated region has been sequences
allele-specific oligonuclotides (ASO)
synthetic prbes that can identify alleles that differ by as little as a single nucleotide
ASOs vs. restriction enzymes
ASOs detect single-nucleotide changes of all types including those that don't affect enzyme cutting sites

inreased resolution & wider application
sickle-cell anemia and other disorders can be screened for using
a combination of ASOs and PCR analysis - this is rapid & highly accurate
how to screen for sickle-cell anemia & other point mutations
Beta-globin gene amplified by PCR, put on filter & hybridized to an ASO synthesized from a normal or mutant copy of the beta-globin gene - normal AA produces a dark spot (2 copies of allele), heterozygous a light spot (one copy), sickle-cell won't bind the probe so see nothing
cystic fibrosis
caused by deletion delta 508 in 70% of all mutant copies of the gene - defect in protin cystic fibrosis transmembrane conducance regulator which regulates chloride ion transport across the plasma membrane