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Slide 1
Gene engineering & Gene expression in vitro
Slide 2
Genentech Founders 1973,Stanley Cohen and Herbert Boyer DNA
recombination pSC101 RSF1010 pSC109 E coli C600
Slide 3
DNA recombination concept: DNA molecule from different scources
covalently ligated with phosphodieaster bond to produce a noval DNA
molecule
Slide 4
Basic concepts zgenetic engineering are terms that apply to the
direct manipulation of an organism's genes. It uses the techniques
of molecular cloning and transformation to alter the structure and
characteristics of genes directly.organismgenesmolecular
cloningtransformation Molecular cloning Molecular cloning refers to
the procedure of isolating a defined DNA sequence and obtaining
multiple copies of it in vivo. Cloning is frequently employed to
amplify DNA fragments containing genes, but it can be used to
amplify any DNA sequence such as promoters, non-coding sequences,
chemically synthesised oligonucleotides and randomly fragmented
DNA.Its core technique is DNA recombination refers to the procedure
of isolating a defined DNA sequence and obtaining multiple copies
of it in vivo. Cloning is frequently employed to amplify DNA
fragments containing genes, but it can be used to amplify any DNA
sequence such as promoters, non-coding sequences, chemically
synthesised oligonucleotides and randomly fragmented DNA.Its core
technique is DNA recombinationin
vivogenespromotersoligonucleotidesin
vivogenespromotersoligonucleotides clone In genetics, an exact
replica of all or part of a macromolecule (e.g. DNA) In cell
biology, a group of identical cells naturally derived from a common
mother cell; of significance in vertebrate physiology, and concepts
related to immunology and cancer biology
Slide 5
Section 1 Tool enzymes zDNA recombination technique is hghly
dependent on tool enzymes and various vectors zTool enzymes:
incision, synthesis, ligation and modification
Slide 6
restriction enzyme zan enzyme that cuts double-stranded or
single stranded DNA at specific recognition nucleotide sequences
known as restriction sites. enzymeDNAnucleotiderestriction
sitesenzymeDNAnucleotiderestriction sites zSuch enzymes, found in
bacteria and archaea, are thought to have evolved to provide a
defense mechanism against invading viruses. Inside a bacterial
host, the restriction enzymes selectively cut up foreign DNA in a
process called restriction; host DNA is methylated by a
modification enzyme (a methylase) to protect it from the
restriction enzymes activity. bacteria archaeaviruses
methylatedmethylasebacteria archaeaviruses methylatedmethylase
zthese two processes form the restriction modification system.
restriction modification system restriction modification
system
Slide 7
1. Classification of restriction enzyme zTypical type II
restriction enzymes differ from type I restriction enzymes in
several ways. They are composed of only one subunit, their
recognition sites are usually undivided and palindromic and 4-8
nucleotides in length, they recognize and cleave DNA at the same
site, and they do not use ATP or AdoMet for their activity they
usually require only Mg2+ as a cofactor.In the 1990s and early
2000s, new enzymes from this family were discovered that did not
follow all the classical criteria of this enzyme class, and new
subfamily nomenclature was developed to divide this large family
into subcategories based on deviations from typical characteristics
of type II enzymes.[15] These subgroups are defined using a letter
suffix.Type II of restriction enzymes are widely used
nomenclature[15] nomenclature[15]
Slide 8
2. Nomenclature of restriction enzyme Each enzyme is named
after the bacterium from which it was isolated using a naming
system based on bacterial genus, species and strain.genus
speciesstrain
Slide 9
3. Characterics of type II restriction enzyme zRecognization
site undivided and palindromic and 4-8 nt in length Rsa I EcoR I
Not I Rsa I EcoR I Not I GTAC GAATTC GCGGCCGC GTAC GAATTC GCGGCCGC
CATG CTTAAG CGCCGGCG CATG CTTAAG CGCCGGCG z6nt palindromic sequence
is widely used the frequency of cutting site: 4 6 4096 bp the
frequency of cutting site: 4 6 4096 bp zSpecial recognization site
BstE II EcoR II BstE II EcoR II GGTNACC CC GG GGTNACC CC GG AT
Slide 10
zCutting modes Sticky end 5 overhang 3 overhang 3 overhang
Blunt end zDNA molecule with same end can be recognized and
ligated
Slide 11
isoschizomer and isoaudamer isoschizomer and isoaudamer
zIsoschizomers are pairs of restriction enzymes specific to the
same recognition sequence. restriction enzymesrecognition
sequencerestriction enzymesrecognition sequence BamH I and BstI G
GATCC BamH I and BstI G GATCC Xho I and PaeR7 C TCGAG Xho I and
PaeR7 C TCGAG zisoaudamer An enzyme that recognizes slightly
different sequence, but produces the same ends BamH I Sau3A I BamH
I Sau3A I GGATCC NGATCN GGATCC NGATCN CCTAGG NCTAGN CCTAGG
NCTAGN
Slide 12
4. Application of restriction enzyme zEssential tool for DNA
recombination yDNA cutting yMapping the physical map of genome
yDetection of DNA mutation (RFLP) zReaction for restriction enzyme
yMg 2+ ypH neutral condition yIonic strength with different
concentration of Na+ or Ka+
Slide 13
Restriction digestion of lamda bateriaophage DNA
Slide 14
modifcation enzyme z 1) DNA polymerase I MW 109KD 5, 3,
polymerizarion 5, 3, polymerizarion y 3, 5, exonuclease activity 5,
3, exonuclease activity 5, 3, exonuclease activity yapplication
nickle translation the second strand synthesis of cDNA, DNA 3end
labeling nickle translation the second strand synthesis of cDNA,
DNA 3end labeling DNA pol I 53
Slide 15
zKlenow fragment 76KD) z5, 3, polymerization and 3, 5,
exonuclease activity yBlunting the 3end yLabeling the 3end
ySynthesis of the second strand of cDNA yDNA sequencing zTaq DNA
polymerase yCloned from Thermus aquaticus yNo proof reading
activity and the optimal temperature is 72 yUsed in PCR Klenow
Slide 16
2 reverse transcriptase zReverse transcriptase RNA-dependent
DNA polymerase, is a DNA polymerase enzyme that transcribes
single-stranded RNA into single-stranded DNA. It also helps in the
formation of a double helix DNA once the RNA has been reverse
transcribed into a single strand cDNA. Normal transcription
involves the synthesis of RNA from DNA; hence, reverse
transcription is the reverse of this DNA
polymeraseenzymetranscribesRNADNA polymeraseenzymetranscribesRNADNA
ztypes AMV and MMLV zactivity zreverse transcriptase RNase H DNA
pol RNase H DNA pol zRNase H deleted zPrimer is needed for RT
Slide 17
3 DNA ligase zDNA ligase is a special type of ligase that can
link together two DNA strands that have double-strand break (a
break in both complementary strands of DNA). The alternative, a
single-strand break, is fixed by a different type of DNA ligase
using the complementary DNA as a template but still requires DNA
ligase to create the final phosphodieaster bond to fully repair the
DNA ligaseDNAligaseDNA zLigase mechanism The mechanism of DNA
ligase is to form two covalent phosphodieaster bond between 3
hydroxyl end of one nucleotide with the 5 phosphate end of another.
ATP is required for the ligase reaction. The mechanism of DNA
ligase is to form two covalent phosphodieaster bond between 3
hydroxyl end of one nucleotide with the 5 phosphate end of another.
ATP is required for the ligase reaction. zLigase works both with
blunt and sticky ends of DNA. T4 DNA Ligase T4 DNA Ligase Ecoli DNA
ligase Ecoli DNA ligase
Slide 18
4 alkaline phosphatase zAlkaline phosphatase is a hydrolase
enzyme responsible for removing phosphate groups from many types of
molecules, including nucleotides, proteins, and alkaloids.
hydrolaseenzymephosphatenucleotidesproteinsalkaloidshydrolaseenzymephosphatenucleotidesproteinsalkaloids
zCalf intestinal alkaline phosphatase (CIAP) Application: prevent
vector self-ligation 5end labeling 5end labeling enzyme-linked
developing enzyme-linked developing
Slide 19
5 Terminal deoxynucleotidyl transferase(TdT) zTdT catalyses the
addition of nucleotides to the 3' terminus of a DNA molecule.
Unlike most DNA polymerases it does not require a template. The
preferred substrate of this enzyme is a 3'-overhang, but it can
also add nucleotides to blunt or recessed 3' ends.
catalysesnucleotides3' terminus DNA
3'-overhangcatalysesnucleotides3' terminus DNA 3'-overhang zHighest
activity with 3end overhang zLabeling of 3end Adding homo-polytail
Adding homo-polytail cpnstructing artifical sticky end cpnstructing
artifical sticky end
Slide 20
Section 2 vector zVector:vehicle used to transfer genetic
material to a target cell zCharacterics of vectors yorgin
ySelection marker yMultiple cloning site yCapacity
Slide 21
Vectors which host is Ecoli plasmid bacteriophage cosmid M13
phage
Slide 22
plasmid zA plasmid is an extra-chromosomal DNA molecule
separate from the chromosomal DNA which is capable of replicating
independently of the chromosomal DNA. In many cases, it is circular
and double-stranded. Plasmids usually occur naturally in bacteria,
but are sometimes found in eukaryotic organisms (e.g., the
2-micrometre-ring in Saccharomyces cerevisiae DNAchromosomal DNA
Saccharomyces cerevisiaeDNAchromosomal DNA Saccharomyces
cerevisiae
Slide 23
Natural plasmid classification yFertility-F-plasmids, which
contain tra-genes. They are capable of conjugation (transfer of
genetic material between bacteria which are touching).
F-plasmidsconjugationF-plasmidsconjugation yResistance-(R)plasmids,
which contain genes that can build a resistance against antibiotics
or poisons and help bacteria produce pili. Historically known as
R-factors, before the nature of plasmids was understood.
antibioticspoisonsantibioticspoisons yCol-plasmids, which contain
genes that code for (determine the production of) bacteriocins,
proteins that can kill other bacteria. bacteriocinsproteins
bacteriocinsproteins Features of Plasmid used in molecular biology
yRelativel small molecula weight yDrug resistance gene yMultiple
cloning site yOrgin site for replication
Slide 24
zApplication of plasmids cloning cloning Sequencing Sequencing
In vitro transcription In vitro transcription Gene expression Gene
expression
Slide 25
Main types of plasmids in molecular biology zpBR322 ymost
commonly used E. coli cloning vectors. E. colicloningvectorsE.
colicloningvectors ythe first artificial plasmid. Created in 1977,
it was named eponymously after its Mexican creators, p standing for
plasmid, and BR for Bolivar and Rodriguez. y4363 base pairs in
length and contains a replicon region (source plasmid pMB1), the
ampR gene, and the tetR gene,(source plasmid pSC101). pSC101 yThe
plasmid has unique restriction sites for more than forty
restriction enzymes. 11 of these 40 sites lie within the tetR gene.
There are 2 sites for HindIII and within the promoter of the tetR
gene. There are 6 key restriction sites inside the ampR gene. The
origin of replication or ori site in this plasmid is pMB1 (a close
relative of ColE1)[2]. The ori encodes two RNAs (RNAI and RNAII)
and one protein (called Rom or Rop). HindIII restriction
sitesori[2]HindIII restriction sitesori[2]
Slide 26
zpUC18 and pUC19 yHigh copies in cell yContaining LacZ to
faciliate the recombinant selection yMCS containing more enzyme
recognization site zpUC118/119 yRepilcation origin based on
pUC18/19 based on pUC18/19
Slide 27
bacteriophage zThe virus particle consists of a head and a tail
that can have tail fibres. The head contains 48,490 base pairs of
double-stranded, linear DNA flanked by 12-base-pair, single-
stranded segments that make up the two strands of the cos site. In
its circular form in the host cytoplasm, the phage genome therefore
is 48,502 base pairs in length. The prophage exists as a linear
section of DNA inserted into the host chromosome zLytic or
lysogenic
Slide 28
Charterictics of phage zBig genome,packing into virus particle
to infect cell zCos site is needed for packaging, recombinant size
ranging between 75 -105% of genome z40 of genome can be replaced.
The longest insert is 23kb
Slide 29
Types of phage zTwo types : yInsetion vector one restricition
enzyme recognization site theoritical capacity 0 13.5 kb
theoritical capacity 0 13.5 kb yReplacement vector two RE
recognization site theoritical capacity 9 22.5 kb theoritical
capacity 9 22.5 kb
Slide 30
Types of phage DNA zCharon 40 replacement, insert 9.2 24.2 kb
zEMBL 3 replacement, insert 7 22 kb Spi selection Spi selection zgt
10 insertion insert 0 7.6 kb zgt 11 insertion insert 0 7.2 kb LacZ
gene selection LacZ gene selection zDASH replacement insert 9 22 kb
Spi selection T3 and T7 promoter Spi selection T3 and T7 promoter
zZAP insertion insert less than10 kb ColE1 replicon convert to
plasmid ColE1 replicon convert to plasmid
Slide 31
cosmid zA cosmid, first described by Collins and Hohn in 1978,
is a type of hybrid plasmid (often used as a cloning vector) that
contains cos sequences, DNA sequences originally from the lambda
phage. Cosmids can be used to build genomic library. Insert 40-45
kb
Slide 32
Features of cosmid zadvantages yAmp r or Tet r yCos site in
vitro packaging yOne or more RE recognization site yHigh capacity
especially for genomic library construct yNon-recombinant cosmid is
small, can not be in vitro packaged, easily for selection
zdisadvantages The length of insert affect the amplification
efficiency
Slide 33
Filamentous phage vector zPhage particle is filamentous,
circular single-strand DNA genome, the length is 6.5kb M13, f1,fd
M13, f1,fd zM13 only infect the Ecoli with F factor
zM13mp18/M13mp19 yContaining LacZ and MCS yLimited packing capacity
used in sequencing
Slide 34
Other vectors YAC capacity 0.5-2MB
Slide 35
BAC capacity 0.1-0.4MB
Slide 36
expressing vector zAn expression vector, otherwise known as an
expression construct, is generally a plasmid that is used to
introduce a specific gene into a target cell. Once the expression
vector is inside the cell, the protein that is encoded by the gene
is produced by the cellular-transcription and translation
machinery.plasmidgeneprotein encodedtranscription translation
zExpression vector is frequently engineered to contain regulatory
sequences that act as enhancer and promoter regions and lead to
efficient transcription of the gene carried on the expression
vector. The goal of a well-designed expression vector is the
production of large amounts of stable messenger
RNAenhancerpromotermessenger RNA zHost cell prokaryote and
eukaryote
Slide 37
expression vector in Ecoli promoter SD sequence terminator
Expression element
Slide 38
Commonly used prokaryotic promoter zTac promoter heterogenous
promoter with Trp (-35), artifical synthesised -10 regions and Lac
operator. Containing LacI heterogenous promoter with Trp (-35),
artifical synthesised -10 regions and Lac operator. Containing LacI
very strong promoter can be induced by IPTG very strong promoter
can be induced by IPTG zT7 phage promoter highly specific promoter
only expressed in T7 RNA polymerase-containing host cell. Lac
operator in the downstream of T7 promoter, and can be induced by
IPTG Lac operator in the downstream of T7 promoter, and can be
induced by IPTG zpL promoter of controlled by temperature-sensitive
inhibitors cIts857 repress transcription at low temprature and
ddoes not work at high temparature
Slide 39
Slide 40
S D sequence zLocated between transcription starting site and
initiation zThe distance between SD sequence and initiation codon
is very important to transcription activiety
Slide 41
transcription termination sequence zlength to 800bp
zcharacterics strong termination for RNA ploymerase, A/T rich and
G/C rich Palindromic structure
Slide 42
Slide 43
expression vector in mammalian zBasic element needed ori,
resistance gene, MCS ori, resistance gene, MCS zElement needed for
expression yPromoter and enhancer from virus, strong activity yPoly
A tailing AAUAAA ySplicing signal provided by insert ySelection
marker tk and neo r
Slide 44
Slide 45
Section 3 Process of gene cloning
Slide 46
preparation of target DNA first and very important step for
cloning first and very important step for cloning Strategy for gene
cloning Strategy for gene cloning positional clonging: RE digestion
positional clonging: RE digestion directional cloning directional
cloning Sequence-based cloning: PCR Sequence-based cloning: PCR
chemical synthesis chemical synthesis capture from library capture
from library Function-based cloning immuno selection
Slide 47
preparation of genomic DNA zgenomic library: a population of
host bacteria, each of which carries a DNA molecule that was
inserted into a cloning vector, such that the collection of cloned
DNA molecule represents the entire genome of the source organism.
This term also represents the collection of all of the vector
molecules, each carrying a piece of the chrosomal DNA of the
organism, prior to the insertion of these molecules into the host
cells. populationDNApopulationDNA zVectors: plasmid, phage, YAC
zshotgun zUse: Sequencing for whole genome
Slide 48
preparation of cDNA zA cDNA library is a collection of cloned
cDNA fragments inserted into a collection of host cells, which
together constitute some portion of the transcriptome of the
organism. cDNA z cDNA is produced from fully transcribed mRNA found
in the nucleus and therefore contains only the expressed genes of
an organism. mRNA
Slide 49
Full length cDNA synthesis zSMART Switch mechanism at RNA
terminal
Slide 50
Characterics of cDNA zExpressed library zLack of intron, and
can be expressed in prokaryote ztissue specific cDNA libraries zLow
abundant mRNA is difficult to get from cDNA library
Slide 51
PCR zPCR polymerase chain reaction zThe simplest way to obtain
the specific DNA
Slide 52
Chemical synthesis zOligonucleotide by chemical synthesis
(poly">
Application of SDM zLong-term insulin chain B Thr27Arg chain A
Asn21Gly C-terminal NH2 result pI 5.4 pI6.8 zRapid absorbe insulin
monomer>polymer zIncreasing the affinity of receptor chain B
His27Asp