140 lec 4th exam reviewer

8/10/2019 140 Lec 4th Exam Reviewer

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Recombinant DNA Technology

Recombinant DNA Genetic Engineering or

cloning DNA molecule formed in the

laboratory by joiningtogether DNA sequencesfrom di erent biologicalsources

Technology that is utilized tocreate and study thesehybrid molecules

DNA USEDo DNA used to multi lyo !arrier"#ector$ used to

carry multi lier DNA

to host cell %here it%ill be e& ressed STE'S

o 'urify DNA to becloned

o (estriction enzymesare used to generates eci)c DNAfragments *recognizeand cut+

o ,ragments joined toother DNA moleculescalled vectors orcarrier molecules

-ector . DNAfragment /recombinantDNA molecule

o (ecombinant DNAmolecule transferredto a host cell

!om etent cellscan ta0e uDNA1

o 2ost cells re licateand ass recombinantDNA to rogeny

!loned DNA canbe reco#eredfrom host cells

!loned DNA canbe transcribed3

m(NAtranslated andthen geneproduct isisolated forresearch

How to Make a Bacterial Cell Competent

Treat with CaCl2: Ca2+neutralizes DNA (which isbasic) and this facilitatesentry

Electroporation: electriccurrent passed throu h host cell open pores andfacilitates DNA entry

!icroin"ection: introductioninto bi cells li#e oocytes(can be seen under a$icroscope)

%ene %un (sa$e as$icroin"ection but usin aun)

Restriction Enzymes 'roduced by bacteria as a

defense mechanism against#iral infection by degrading

the DNA of in#ading #iruses*cutting foreign DNA+

o 425 D6N7T T2E5 !UT8A!TE(9A: DNA;(estriction sites aremethylated and arenot recognized

'(6!ESSo (ecognizes s eci)c

nucleotide sequencecalled recognitionsequences

o !uts both strands of DNA %ithin thesequence

o < n / number of baseairs to be cut ona#erage *n /nucleotides it canrecognize+



!uts DNA at s eci)crecognitions silence

'alindromic sequence Stic0y ends$ un aired after

cutting3 so the sequence has

otential to base air Stic0y ends of DNA of interest is madecom lementary to lasmidby cutting it %ith the sameendonuclease

:igase seals the ga s

Vectors• Transfer and hel re licate

inserted DNA fragments• Di erences 8et%een -ectors

o 2osts they can entero Size of inserts they

can carryo Number of co ies

roducedo Number of recognition

sequences a#ailableo Number and ty e of

selectable mar0ergenes

• 'ro erites of a -ectoro

!an re licateinde endently along%ith any DNAfragment it carriesonce inside the hostcell

6rigin of re lication*initiation+

o !ontain se#eralrestriction enzymeclea#age sites that

allo% insertion of DNAfragments to becloned

o !arry a selectablemar0er gene toidentify host cells thatcontain recombinant#ectors

o 11DNA should be easyto reco#er from hostcell

• (ecombinant -ector$ #ector%ith an inserted fragment

• 'lasmidso Genetically modi)ed

lasmids %ere the )rst#ectors de#elo ed

o Small3 easy to use andcan re roduceinde endently

o E&trachromosomaldouble=stranded DNAmolecule thatre licatesautonomously in

bacterial cells>circular> strong originof re lications> notessential but hasselecti#e ad#antage*not resent in all+

o 9m ortance in8acteria$ genesresistant to antibiotics

o p&C's$all (2base pairs( with

lar e DNAinsertsori in of replicationlar e nu$ber of restrictionenzy$ereco nitionse*uences in

polylin#er siteeasily identi ed: bluecolonies in , al(can $etabolizeas induced by -.T%)

• :ambda 'hageo !entral gene cluster

can be remo#ed and



re laced %ithrecombinant DNA

o !an carry u to ?@0bof cloned DNA

• !osmidso :ambda hage .

'lasmidso !ontain cos sites of

lambda hage forac0aging hage DNAinto hage articles

o 9nside the cell3 itre licates li0elasmids

o !an carry u to @0bof inserted DNA

• S ecialized -ectorso E& ression #ectors$engineered %ith

control sequences thatallo% e& ression of inserted genes

o 8acterial Arti)cial!hromosomes *8A!s+and 5east Arti)cial!hromosomes *5A!s+$designed to clone #erylong segments of DNA

Types of Bacterial Cells and What to Eliminate

/,0 1ithout plas$ido use a$picillin

/,01ith plas$id but not reco$binant

o absence of lac reco$binant then thiswill produce bluecolonies

1ith plas$id andreco$binant

o -n the presence of lac reco$binant3 will

produce whitecolonies

Transgenic Plant 'lant %ith DNA from other

source

A robacteriu$ tu$efaciens $naturally infecting lant cells

Ti lasmid$ tumor inducinglasmid T=DNA cro%n

gall or tumor Bar0er$ 0an ( / 0anomycin

resistant

Recombinant Libraries• DNA library$ a set of DNA

clones deri#ed from a singlesource

• Genomic library$ contains atleast one co y of e#erysequence in an organism7sgenome

o !onstructed using

host=cell cloningmethods

• !hromosome s eci)c libraryo !onstructed using

Co% cytometry• cDNA library$ contains DNA

co ied from messenger (NAmolecules resent in a cello ulation at a gi#en time>

genes being e& ressed>isolate m(NA not genomicDNA *has introns+

o 11BA 9NG T2E !DNA:98(A(5 :9N E(SEFUEN!ES

o !an be constructedusing (T='!( *re#ersetrancri tase '!(+ orcon#entional cDNAre aration

• Screening a :ibrary$ NucleicAcid 2ybridization

o 'robe$ any DNA or

(NA sequence thathas been labeled insome %ay and iscom lementary tosome art of a clonedsequence resent inthe library

(adioacti#e'hos horus or



enzymes %ithcoloredchemicalroduct11'(6!ESS11:98(A(5 6,'2AGE !:6NES

Amplifying DNA P!R (a id method of co ying

DNA that e&tends the o%erof recombinant DNAresearch ad eliminates theneed to use host cells forcloning

Ste so 2eatingo

!oolingo (e lication

ey$ heat=stable DNAolymerase *Taq olymerasefrom Ther$ophilusa*uaticus +

(equirementso Single stranded DNA

tem lateso 'rimers for each

strand T%o rimers

o DNA olymerase Bg ?. is a

cofactor 11'(6!ESS

Cloning Animals• Nuclear Transplantation

o Nucleus of anunfertilized e isreplaced with thenucleus of a

di4erentiated cell(e5 5 fro e$bryos)• 6eproducti7e Clonin of

!a$$also Dolly the 8heep

Nucleus fro$$a$$ary land cell(contains entire

eno$e)9cloned becausee actly thesa$e DNA

"ene #utations and Repair

"ene #utations !hange in nucleotide

sequence 6rigin of genetic #ariation

%ithin o ulations S ontaneous #s 9nduced

o S ontaneous$ changesin the nucleotidesequence of genesthat a ear to ha#e

no 0no%n causeo 9nduced$ mutationsthat result from theinCuence of e&traneous factors*natural or arti)cial+

8ased on :ocationo Somatic$ occurring in

any cell in the bodye&ce t germ cells> notheritable

o Germline$ occurring in

gametes> heritableo Autosomal$ mutations

%ithin genes locatedon the autosomes

o H=lin0ed$ %ithin geneslocated on the H=chromosome

8ased on 'henoty ic E ecto :oss of function$

reduces or eliminatesthe function of a generoduct

o Gain of function$ generoduct %ithenhanced or ne%functions> mostlydominant

o Bor hological"-isible$a ecting amor hological trait



o Nutritionalo 8iochemical

Sic0le=cellanemia hemo hilia

o 8eha#ioralo (egulatory mutationso :ethal$ interru t a

rocess that isessential to thesur#i#al of theorganism

o !onditional$e& ression de endson the en#ironment in%hich the organism)nds itself

Tem erature=sensiti#emutations$ermissi#e andrestricti#etem eratures

o Neutral$ mutations areli0ely to occur in thelarge ortions of thegenome that do notcontain genes>majority of mutations

8ased on Ty e of Bolecular!hange

o 'oint Butation or 8aseSubstitution

Bissensemutation$ ne%tri let code thatcodes for adi erent aminoacid

Nonsensemutation$changed into asto codon

Silent mutation$alters a codonbut doesnotresult in a

change in theamino acid

Transition$yrimidinere laces ayrimidine orurine re lacesurine

Trans#ersion$urine re lacesyrimidine or#ice #ersa

o ,rameshift Butations :oss or addition

of a singlenucleotidecauses all of thesubsequentthree=lettercodons to bechanged*addition3 ordeletion+

S ontaneous Butations$(e lication Errors and 8aseBodi)cations

o DNA (e lication Errors Usually

corrected by

DNAolymeraseo (e lication Sli age

m(NA$ shorter tem late$

longer 6ccurs

any%here inDNA but morecommon inregions %ithre eatedsequences

!auses$ onestrand of DNAtem late loo sout andbecomesdis laced> DNA



olymerasesli s or stutters

o Tautomeric Shifts 8ases can ta0e

se#eral forms*tautomers+increaseschange of mis airing

Amino iminoo De urination and

Deamination A urinic sites$

randomlacement by

DNAolymerase

De urination$loss of one of the nitrogenousbases in anintact double=helical DNAmolecule

Deamination$an amino grouin cytosine oradenine iscon#erted to a

0eto grou !ytosine

uracil Adenine

hy o&anthine

o 6&idati#e Damage Su ero&ides3

hydro&ylradicals3ero&ide

o Trans osons DNA elements

that can mo#e%ithin orbet%eengenomes

Naturallyoccurringmutagens

Trans osasegene$ allo%s tomo#e todi erentlocations>

jum inggenes"sel)shgenes

Nonre licati#e (e licati#e 11!6B'6S9TE

AND S9B':E T(ANS'6S6N

9nduced Butations

o 8ase Analog Substitutes realbases duringnucleic acidbiosynthesis

o Al0ylating Agents Donate an al0yl

grou to aminoor 0eto grou sin nucleotides

o 9ntercalating Agents I rings can )t

%here basesare

o U- (adiationo 9onizing (adiation

Strand brea0s

"ene Repair 'roofreading

o DNA 'olymerase 999 E&onuclease

acti#ity *I7

7+o 9f an incorrectnucleotide is insertedduring olymerization3the enzyme canrecognize the errorand re#erse itsdirection

Bismatch (e air



o After roofreadingo Strand Discrimination

Adeninemethylase

Bethylated istem late DNA

o There are ?e&onucleasesde ending ondirection * 7 I7 or I7

7+o 9ncorrect nucleotide

remo#ed3 correctnucleotide re laces it

o (e air enzyme bindsto unmethylated DNA

endonuclease

creates nic0 in thebac0bonee&onuclease un%indsand degrades DNAuntil region of mismatched isreached DNAolymerase )lls in thega %ith correctnucleotide DNAligase seals the ga

'ostre lication (e airo ,or thymine dimerso (es onds after

damaged DNA hasesca ed re air andhas failed to becom letely re licated

o Thymine dimer stillthere but %ithre lication instead of none at all

o (ecA res onsible forrecombination %ithcom lementary region

S6S (es onseo 9n bacteria *le&A3 recA

and u#r+ can allo%DNA re lication tooccur e#en in theresence of lesions

o (ecA co roteaseremo#e le&A (NAolymerase acti#atesre air mechanism

o Error roneo ,or thymine dimer

• 'hotoreacti#ation (e airo ,or thymine dimero (eal re airo !lea#es bonds

bet%een thyminedimers and directlyre#erses the e ect of U- radiation in DNA

• Mechanism for ExcisionRepair

o Error is reco nized

and enzy$atically clipped out by anendonuclease

o DNA poly$erase llsthe ap9 Adds to ;<=>? so usually done by DNA poly$erase -

o DNA li ase seals thenic#

• 8ase E&cision (e airo DNA glycosylase$

s eci)c for base> cutsthe glycosidic bondbet%een base andsugar

o A' endonuclease$recognizes sugar %ithmissing bases

o Endonucleasesremo#e deo&yribosesugar DNAolymerase DNAligase

o !orrects DNA thatcontains a damagedDNA base

• Nucleotide E&cision (e airo 8igger co#erage than

base e&cision re airo (e air bul0y lesions in

DNA that alter or



distort the doubleheli&

o 'olymerase 9$ shorterbases re licated thanolymerase 999

o Herodermaigmentosum

• 2omologous (ecombination(e air

o 'ostre lication re air

o 9onizing radiationo Usually occurs during

the late S or early G?hase *sisterchromatids area#ailable+

o Accurate rocessbecause anundamaged tem lateis used

140 lec 4th exam reviewer

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