biochemistry finals

8/17/2019 Biochemistry Finals

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No. 38Policarpio, Anna Francheska G.2E – Pharmacy

ASSIGNMENT IN I!"#EMIST$%

RECOMBINANT DNA TECHNOLOGY

$ecom&inan' (NA Technolo)y is 'he *oinin) 'o)e'her o+ (NA molecles +rom '-o

i++eren' species 'ha' are inser'e in'o a hos' or)anism 'o proce ne- )ene'ic

com&ina'ions 'ha' are o+ /ale 'o science, meicine, a)ricl're, an ins'ry. $0(NA

'echnolo)y ses palinromic se1ences an leas 'o 'he proc'ion o+ s'icky an &ln'

ens.

Since 'he +ocs o+ all )ene'ics is 'he )ene, 'he +namen'al )oal o+ la&ora'ory

)ene'icis's is 'o isola'e, charac'erie, an manipla'e )enes. Al'ho)h i' is rela'i/ely

easy 'o isola'e a sample o+ (NA +rom a collec'ion o+ cells, +inin) a speci+ic )ene -i'hin

'his (NA sample can &e compare 'o +inin) a neele in a hays'ack. "onsier 'he +ac'

'ha' each hman cell con'ains approima'ely 2 me'res 45 +ee'6 o+ (NA. There+ore, a

small 'isse sample -ill con'ain many kilome'res o+ (NA. #o-e/er, recom&inan' (NA

'echnolo)y has mae i' possi&le 'o isola'e one )ene or any o'her se)men' o+ (NA,

ena&lin) researchers 'o e'ermine i's ncleo'ie se1ence, s'y i's 'ranscrip's, m'a'ei' in hi)hly speci+ic -ays, an reinser' 'he moi+ie se1ence in'o a li/in) or)anism.

APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY

$ecom&inan' (NA is -iely se in &io'echnolo)y, meicine an research. Toay,

recom&inan' pro'eins an o'her proc's 'ha' resl' +rom 'he se o+ r(NA 'echnolo)y

are +on in essen'ially e/ery -es'ern pharmacy, oc'or7s or /e'erinarian7s o++ice,

meical 'es'in) la&ora'ory, an &iolo)ical research la&ora'ory. In ai'ion, or)anisms'ha' ha/e &een manipla'e sin) recom&inan' (NA 'echnolo)y, as -ell as proc's

eri/e +rom 'hose or)anisms, ha/e +on 'heir -ay in'o many +arms, spermarke's,

home meicine ca&ine's, an e/en pe' shops, sch as 'hose 'ha' sell GloFish an o'her

)ene'ically moi+ie animals.

http://www.britannica.com/science/DNAhttp://www.britannica.com/science/species-taxonhttp://www.britannica.com/topic/sciencehttp://www.britannica.com/topic/medicinehttps://en.wikipedia.org/wiki/Sticky_and_blunt_endshttps://en.wikipedia.org/wiki/Sticky_and_blunt_endshttp://www.britannica.com/science/geneticshttp://www.britannica.com/science/genehttp://www.britannica.com/science/DNAhttp://www.britannica.com/science/cell-biologyhttp://www.britannica.com/science/nucleotidehttps://en.wikipedia.org/wiki/Biotechnologyhttps://en.wikipedia.org/wiki/Medicinehttps://en.wikipedia.org/wiki/Researchhttps://en.wikipedia.org/wiki/Genetically_modified_foodhttps://en.wikipedia.org/wiki/Humulinhttps://en.wikipedia.org/wiki/GloFishhttp://www.britannica.com/science/species-taxonhttp://www.britannica.com/topic/sciencehttp://www.britannica.com/topic/medicinehttps://en.wikipedia.org/wiki/Sticky_and_blunt_endshttps://en.wikipedia.org/wiki/Sticky_and_blunt_endshttp://www.britannica.com/science/geneticshttp://www.britannica.com/science/genehttp://www.britannica.com/science/DNAhttp://www.britannica.com/science/cell-biologyhttp://www.britannica.com/science/nucleotidehttps://en.wikipedia.org/wiki/Biotechnologyhttps://en.wikipedia.org/wiki/Medicinehttps://en.wikipedia.org/wiki/Researchhttps://en.wikipedia.org/wiki/Genetically_modified_foodhttps://en.wikipedia.org/wiki/Humulinhttps://en.wikipedia.org/wiki/GloFishhttp://www.britannica.com/science/DNA


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The mos' common applica'ion o+ recom&inan' (NA is in &asic research, in -hich 'he

'echnolo)y is impor'an' 'o mos' crren' -ork in 'he &iolo)ical an &iomeical

sciences. $ecom&inan' (NA is se 'o ien'i+y, map an se1ence )enes, an 'o

e'ermine 'heir +nc'ion. r(NA pro&es are employe in analyin) )ene epression

-i'hin ini/ial cells, an 'hro)ho' 'he 'isses o+ -hole or)anisms. $ecom&inan'

pro'eins are -iely se as rea)en's in la&ora'ory eperimen's an 'o )enera'e

an'i&oy pro&es +or eaminin) pro'ein syn'hesis -i'hin cells an or)anisms.

Many ai'ional prac'ical applica'ions o+ recom&inan' (NA are +on in ins'ry, +oo

proc'ion, hman an /e'erinary meicine, a)ricl're, an &ioen)ineerin).

!'her applica'ions o+ recom&inan' (NA

9. $E"!MINANT "#%M!SIN

• Fon in renne', chymosin is an enyme re1ire 'o man+ac're cheese. I' -as

'he +irs' )ene'ically en)ineere +oo ai'i/e se commercially. Trai'ionally,

processors o&'aine chymosin +rom renne', a prepara'ion eri/e +rom 'he +or'h

s'omach o+ milk0+e cal/es. Scien'is's en)ineere a non0pa'ho)enic s'rain 4:0926

o+ E. coli &ac'eria +or lar)e0scale la&ora'ory proc'ion o+ 'he enyme. This

micro&iolo)ically proce recom&inan' enyme, ien'ical s'rc'rally 'o 'he cal+

eri/e enyme, cos's less an is proce in a&nan' 1an'i'ies. Toay a&o'

5;< o+ =.S. har cheese is mae -i'h )ene'ically en)ineere chymosin. In 9>>;,

F(A )ran'e chymosin ?)enerally0reco)nie0as0sa+e? 4G$AS6 s'a's &ase on

a'a sho-in) 'ha' 'he enyme -as sa+e.

2. $E"!MINANT #=MAN INS=@IN

• Almos' comple'ely replace inslin o&'aine +rom animal sorces 4e.). pi)s an

ca''le6 +or 'he 'rea'men' o+ inslin0epenen' ia&e'es. A /arie'y o+ i++eren'

recom&inan' inslin prepara'ions are in -iesprea se. $ecom&inan' inslin is

syn'hesie &y inser'in) 'he hman inslin )ene in'o E. coli , or yeas'

4saccharomyces cere/isiae6, -hich 'hen proces inslin +or hman se.

3. $E"!MINANT #=MAN G$!T# #!$M!NE

• Aminis'ere 'o pa'ien's -hose pi'i'ary )lans )enera'e ins++icien' 1an'i'ies 'o

sppor' normal )ro-'h an e/elopmen'. e+ore recom&inan' #G# &ecame

a/aila&le, #G# +or 'herape'ic se -as o&'aine +rom pi'i'ary )lans o+

caa/ers. This nsa+e prac'ice le 'o some pa'ien's e/elopin) "re'+el'–

Bako& isease. $ecom&inan' #G# elimina'e 'his pro&lem, an is no- se

https://en.wikipedia.org/wiki/Rennethttps://en.wikipedia.org/wiki/Diabeteshttps://en.wikipedia.org/wiki/Diabeteshttps://en.wikipedia.org/wiki/E._colihttps://en.wikipedia.org/wiki/Creutzfeldt%E2%80%93Jakob_diseasehttps://en.wikipedia.org/wiki/Creutzfeldt%E2%80%93Jakob_diseasehttps://en.wikipedia.org/wiki/Rennethttps://en.wikipedia.org/wiki/Diabeteshttps://en.wikipedia.org/wiki/E._colihttps://en.wikipedia.org/wiki/Creutzfeldt%E2%80%93Jakob_diseasehttps://en.wikipedia.org/wiki/Creutzfeldt%E2%80%93Jakob_disease


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'herape'ically. I' has also &een misse as a per+ormance enhancin) r) &y

a'hle'es an o'hers.

4. $E"!MINANT @!!( "@!TTING FA"T!$ CIII

A &loo0clo''in) pro'ein 'ha' is aminis'ere 'o pa'ien's -i'h +orms o+ 'he &leein)

isorer hemophilia, -ho are na&le 'o proce +ac'or CIII in 1an'i'ies s++icien'

'o sppor' normal &loo coa)la'ion. e+ore 'he e/elopmen' o+ recom&inan'

+ac'or CIII, 'he pro'ein -as o&'aine &y processin) lar)e 1an'i'ies o+ hman

&loo +rom ml'iple onors, -hich carrie a /ery hi)h risk o+ 'ransmission

o+ &loo &orne in+ec'ios iseases, +or eample #IC an hepa'i'is .

5. $E"!MINANT #EPATITIS CA""INE

#epa'i'is in+ec'ion is con'rolle 'hro)h 'he se o+ a recom&inan' hepa'i'is

/accine, -hich con'ains a +orm o+ 'he hepa'i'is /irs sr+ace an'i)en 'ha' is

proce in yeas' cells. The e/elopmen' o+ 'he recom&inan' s&ni' /accine

-as an impor'an' an necessary e/elopmen' &ecase hepa'i'is /irs, nlike

o'her common /irses sch as polio /irs, canno' &e )ro-n in /i'ro.

5. (IAGN!SIS !F INFE"TI!N IT# #IC

• Each o+ 'he 'hree -iely se me'hos +or ia)nosin) #IC in+ec'ion has &een

e/elope sin) recom&inan' (NA. The an'i&oy 'es' 4E@ISA or -es'ern &lo'6

ses a recom&inan' #IC pro'ein 'o 'es' +or 'he presence o+ an'i&oies 'ha' 'he

&oy has proce in response 'o an #IC in+ec'ion. The (NA 'es' looks +or 'he

presence o+ #IC )ene'ic ma'erial sin) re/erse 'ranscrip'ion polymerase chainreac'ion 4$T0P"$6. (e/elopmen' o+ 'he $T0P"$ 'es' -as mae possi&le &y 'he

moleclar clonin) an se1ence analysis o+ #IC )enomes.

7. G!@(EN $I"E

• A recom&inan' /arie'y o+ rice 'ha' has &een en)ineere 'o epress 'he enymes

responsi&le +or D0caro'ene &iosyn'hesis. This /arie'y o+ rice hols s&s'an'ial

promise +or recin) 'he incience o+ /i'amin A e+iciency in 'he -orl7s

popla'ion. Golen rice is no' crren'ly in se, penin) 'he resol'ion o+

re)la'ory an in'ellec'al proper'y isses.

8. #E$I"I(E0$ESISTANT "$!PS

• "ommercial /arie'ies o+ impor'an' a)ricl'ral crops 4inclin) soy, maiecorn,

sor)hm, canola, al+al+a an co''on6 ha/e &een e/elope 'ha' incorpora'e a

recom&inan' )ene 'ha' resl's in resis'ance 'o 'he her&icie )lyphosa'e 4'rae

https://en.wikipedia.org/wiki/Blood-borne_diseasehttps://en.wikipedia.org/wiki/Hepatitis_Bhttps://en.wikipedia.org/wiki/Polio_virushttps://en.wikipedia.org/wiki/Polio_virushttps://en.wikipedia.org/wiki/In_vitrohttps://en.wikipedia.org/wiki/HIV_testhttps://en.wikipedia.org/wiki/ELISAhttps://en.wikipedia.org/wiki/Western_blothttps://en.wikipedia.org/wiki/Antibodieshttps://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reactionhttps://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reactionhttps://en.wikipedia.org/wiki/%CE%92-carotenehttps://en.wikipedia.org/wiki/Vitamin_A_deficiencyhttps://en.wikipedia.org/wiki/Glyphosatehttps://en.wikipedia.org/wiki/Blood-borne_diseasehttps://en.wikipedia.org/wiki/Hepatitis_Bhttps://en.wikipedia.org/wiki/Polio_virushttps://en.wikipedia.org/wiki/In_vitrohttps://en.wikipedia.org/wiki/HIV_testhttps://en.wikipedia.org/wiki/ELISAhttps://en.wikipedia.org/wiki/Western_blothttps://en.wikipedia.org/wiki/Antibodieshttps://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reactionhttps://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reactionhttps://en.wikipedia.org/wiki/%CE%92-carotenehttps://en.wikipedia.org/wiki/Vitamin_A_deficiencyhttps://en.wikipedia.org/wiki/Glyphosate


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name Roundup6, an simpli+ies -ee con'rol &y )lyphosa'e applica'ion. These

crops are in common commercial se in se/eral con'ries.

9. INSE"T$ESISTANT "$!PS

• Bacillus thuringeiensis is a &ac'erim 'ha' na'rally proces a pro'ein 4' 'oin6

-i'h insec'icial proper'ies. The &ac'erim has &een applie 'o crops as an

insec'0con'rol s'ra'e)y +or many years, an 'his prac'ice has &een -iely aop'e

in a)ricl're an )arenin). $ecen'ly, plan's ha/e &een e/elope 'ha' epress

a recom&inan' +orm o+ 'he &ac'erial pro'ein, -hich may e++ec'i/ely con'rol some

insec' prea'ors. En/ironmen'al isses associa'e -i'h 'he se o+

'hese 'rans)enic crops ha/e no' &een +lly resol/e.

• RESTRICTION

@e' s ne' consier a hyroly'ic reac'ion 'ha' resl's in 'he clea/a)e o+ (NA.

ac'eria an archaea ha/e e/ol/e mechanisms 'o pro'ec' 'hemsel/es +rom /iral

in+ec'ions. Many /irses in*ec' 'heir (NA )enomes in'o cells once insie, 'he /iral (NA

hi*acks 'he cellHs machinery 'o ri/e 'he proc'ion o+ /iral pro'eins an, e/en'ally, o+

pro)eny /irs. !+'en, a /iral in+ec'ion resl's in 'he ea'h o+ 'he hos'. A ma*or pro'ec'i/e

s'ra'e)y +or 'he hos' is 'o se res'ric'ion enoncleases 4res'ric'ion enymes6 'o

e)rae 'he /iral (NA on i's in'roc'ion in'o a cell. These enymes reco)nie

par'iclar &ase se1ences, calle reco)ni'ion se1ences or reco)ni'ion si'es, in 'heir

'ar)e' (NA an clea/e 'ha' (NA a' e+ine posi'ions. The mos' -ell s'ie class o+ res'ric'ion enymes comprises 'he so0calle 'ype II res'ric'ion enymes, -hich clea/e

(NA -i'hin 'heir reco)ni'ion se1ences. !'her 'ypes o+ res'ric'ion enymes clea/e (NA

a' posi'ions some-ha' is'an' +rom 'heir reco)ni'ion si'es.

$es'ric'ion enoncleases ms' sho- 'remenos speci+ici'y a' '-o le/els. Firs',

'hey ms' clea/e only (NA molecles 'ha' con'ain reco)ni'ion si'es 4herea+'er re+erre

'o as co)na'e (NA6 -i'ho' clea/in) (NA molecles 'ha' lack 'hese si'es. Sppose 'ha'

a reco)ni'ion se1ence is si &ase pairs lon). ecase 'here are 5, or ;>5,

se1ences ha/in) si &ase pairs, 'he concen'ra'ion o+ si'es 'ha' ms' no' &e clea/e -ill

&e approima'ely J;;;0+ol as hi)h as 'he concen'ra'ion o+ si'es 'ha' shol &e clea/e.Ths, 'o keep +rom ama)in) hos'0cell (NA, enoncleases ms' clea/e co)na'e (NA

molecles mch more 'han J;;; 'imes as e++icien'ly as 'hey clea/e nonspeci+ic si'es.

Secon, res'ric'ion enymes ms' no' e)rae 'he hos' (NA. #o- o 'hese enymes

mana)e 'o e)rae /iral (NA -hile sparin) 'heir o-nK

https://en.wikipedia.org/wiki/Bt_toxinhttps://en.wikipedia.org/wiki/Transgenichttp://www.ncbi.nlm.nih.gov/books/n/stryer/A5607/def-item/A5630/http://www.ncbi.nlm.nih.gov/books/n/stryer/A5607/def-item/A5608/http://www.ncbi.nlm.nih.gov/books/n/stryer/A5607/def-item/A5630/https://en.wikipedia.org/wiki/Bt_toxinhttps://en.wikipedia.org/wiki/Transgenichttp://www.ncbi.nlm.nih.gov/books/n/stryer/A5607/def-item/A5630/http://www.ncbi.nlm.nih.gov/books/n/stryer/A5607/def-item/A5608/http://www.ncbi.nlm.nih.gov/books/n/stryer/A5607/def-item/A5630/


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The res'ric'ion enonclease Eco$C 4+rom E. coli6 clea/es o&le0s'rane

/iral (NA molecles 'ha' con'ain 'he se1ence JL0GATAT"03L &' lea/es in'ac' hos'

(NA con'ainin) hnres o+ sch se1ences. The hos' (NA is pro'ec'e &y o'her

enymes calle me'hylases, -hich me'hyla'e aenine &ases -i'hin hos' reco)ni'ion

se1ences. For each res'ric'ion enonclease, 'he hos' cell proces a corresponin)

me'hylase 'ha' marks 'he hos' (NA an pre/en's i's e)raa'ion. These pairs o+

enymes are re+erre 'o as res'ric'ion0moi+ica'ion sys'em. e shall re'rn 'o 'he

mechanism se 'o achie/e 'he necessary le/els o+ speci+ici'y a+'er consierin) 'he

chemis'ry o+ 'he clea/a)e process.

GENE TRANSFER

#orion'al )ene 'rans+er, also kno-n as la'eral )ene 'rans+er, 'he 'ransmission

o+ (NA 4eoyri&oncleic aci6 &e'-een i++eren' )enomes. #orion'al )ene 'rans+er is

kno-n 'o occr &e'-een i++eren' species, sch as &e'-een prokaryo'es 4or)anisms

-hose cells lack a e+ine ncles6 an ekaryo'es 4or)anisms -hose cells con'ain a

e+ine ncles6, an &e'-een 'he 'hree (NA0con'ainin) or)anelles o+ ekaryo'es'he

ncles, 'he mi'ochonrion, an 'he chloroplas'. Ac1isi'ion o+ (NA 'hro)h horion'al

)ene 'rans+er is is'in)ishe +rom 'he 'ransmission o+ )ene'ic ma'erial +rom paren's 'o

o++sprin) rin) reproc'ion, -hich is kno-n as /er'ical )ene 'rans+er .

#orion'al )ene 'rans+er is mae possi&le in lar)e par' &y 'he eis'ence o+ mo&ile

)ene'ic elemen's, sch as plasmis 4e'ra chromosomal )ene'icma'erial6, 'ransposons 4*mpin) )enesO6, an &ac'eria0in+ec'in) /irses

4&ac'eriopha)es6. These elemen's are 'rans+erre &e'-een or)anisms 'hro)h i++eren'

mechanisms, -hich in prokaryo'es incle 'rans+orma'ion, con*)a'ion,

an 'ransc'ion. In 'rans+orma'ion, prokaryo'es 'ake p +ree +ra)men's o+ (NA, o+'en in

'he +orm o+ plasmis, +on in 'heir en/ironmen'. In con*)a'ion, )ene'ic ma'erial is

echan)e rin) a 'emporary nion &e'-een '-o cells, -hich may en'ail 'he 'rans+er o+

a plasmi or 'ransposon. In 'ransc'ion, (NA is 'ransmi''e +rom one cell 'o ano'her /ia

a &ac'eriopha)e.

In horion'al )ene 'rans+er, ne-ly ac1ire (NA is incorpora'e in'o 'he )enome o+

'he recipien' 'hro)h ei'her recom&ina'ion or inser'ion. $ecom&ina'ion essen'ially is 'he

re)ropin) o+ )enes, sch 'ha' na'i/e an +orei)n 4ne-6 (NA se)men's 'ha' are

homolo)os are ei'e an com&ine. Inser'ion occrs -hen 'he +orei)n (NA

http://www.ncbi.nlm.nih.gov/books/n/stryer/A5607/def-item/A5630/http://www.britannica.com/science/DNAhttp://www.britannica.com/science/genehttp://www.britannica.com/science/prokaryotehttp://www.britannica.com/science/nucleus-biologyhttp://www.britannica.com/science/eukaryotehttp://www.britannica.com/science/mitochondrionhttp://www.britannica.com/science/chloroplasthttp://www.britannica.com/science/vertical-gene-transferhttp://www.britannica.com/science/genehttp://www.britannica.com/science/plasmidhttp://www.britannica.com/science/transposonhttp://www.britannica.com/science/bacteriophagehttp://www.britannica.com/science/transformation-biologyhttp://www.britannica.com/science/conjugation-sexual-processhttp://www.britannica.com/science/transduction-microbiologyhttp://www.britannica.com/science/recombination-geneticshttp://www.ncbi.nlm.nih.gov/books/n/stryer/A5607/def-item/A5630/http://www.britannica.com/science/DNAhttp://www.britannica.com/science/genehttp://www.britannica.com/science/prokaryotehttp://www.britannica.com/science/nucleus-biologyhttp://www.britannica.com/science/eukaryotehttp://www.britannica.com/science/mitochondrionhttp://www.britannica.com/science/chloroplasthttp://www.britannica.com/science/vertical-gene-transferhttp://www.britannica.com/science/genehttp://www.britannica.com/science/plasmidhttp://www.britannica.com/science/transposonhttp://www.britannica.com/science/bacteriophagehttp://www.britannica.com/science/transformation-biologyhttp://www.britannica.com/science/conjugation-sexual-processhttp://www.britannica.com/science/transduction-microbiologyhttp://www.britannica.com/science/recombination-genetics


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in'roce in'o a cell shares no homolo)y -i'h eis'in) (NA. In 'his case, 'he ne-

)ene'ic ma'erial is em&ee &e'-een eis'in) )enes in 'he recipien'Hs )enome.

"ompare -i'h prokaryo'es, 'he process o+ horion'al )ene 'rans+er in ekaryo'es ismch more comple, mainly &ecase ac1ire (NA ms' pass 'hro)h &o'h 'he o'er

cell mem&rane an 'he nclear mem&rane 'o reach 'he ekaryo'eHs )enome.

S&celllar sor'in) an si)nalin) pa'h-ays play a cen'ral role in 'he 'ranspor' o+ (NA 'o

'he )enome.

Prokaryo'es can echan)e (NA -i'h ekaryo'es, al'ho)h 'he mechanisms &ehin

'his process are no' -ell ners'oo. Sspec'e mechanisms incle con*)a'ion

anenocy'osis, sch as -hen a ekaryo'ic cell en)l+s a prokaryo'ic cell an )a'hers i'

in'o a special mem&rane0&on /esicle +or e)raa'ion. I' is 'ho)h' 'ha' in rare

ins'ances in enocy'osis, )enes escape +rom prokaryo'es rin) e)raa'ion an are

s&se1en'ly incorpora'e in'o 'he ekaryo'eHs )enome.

#orion'al )ene 'rans+er plays an impor'an' role in aap'a'ion an e/ol'ion in &o'h

prokaryo'es an ekaryo'es. For eample, 'he 'rans+er o+ a )ene encoin) a ni1e

me'a&olic enyme +rom a species o+ Pasteurella &ac'eria 'o 'he pro'ooan parasi'e

Trichomonas vaginalis is sspec'e 'o ha/e +acili'a'e 'he la''er or)anismHs aap'a'ion

'o i's animal hos's. @ike-ise, 'he echan)e o+ a )ene +rom a hman cell 'o 'he

&ac'erim Neisseria )onorrhoeaea 'rans+er 'ha' appears 'o ha/e occrre rela'i/ely

recen'ly in 'he &ac'erimHs e/ol'ionmay ha/e ena&le 'he or)anism 'o aap' an

sr/i/e in hmans. Scien'is's ha/e propose 'oo 'ha' 'he recen' e/ol'ion o+ 'he me'hyl

aspar'a'e pa'h-ay o+ me'a&olism in 'he halophilic 4sal'0lo/in)6 archaean #aloarcla

marismor'i ori)ina'e -i'h 'he or)anismHs ac1isi'ion o+ a specialie se' o+ )enes /ia

horion'al 'rans+er.

STICKY ENDS AND BLUNT ENDS

(NA en or s'icky en re+ers 'o 'he proper'ies o+ 'he en o+ a molecle o+ (NA or a

recom&inan' (NA molecle. The concep' is impor'an' in moleclar &iolo)y, especially

http://www.britannica.com/science/eukaryotehttp://www.britannica.com/science/endocytosishttp://www.britannica.com/science/adaptation-biology-and-physiologyhttp://www.britannica.com/science/evolution-scientific-theoryhttp://www.britannica.com/science/protozoanhttp://www.britannica.com/science/endocytosishttp://www.britannica.com/science/gonococcushttp://www.britannica.com/science/metabolismhttp://www.britannica.com/science/Haloarcula-marismortuihttp://www.britannica.com/science/Haloarcula-marismortuihttp://www.britannica.com/science/Haloarcula-marismortuihttps://en.wikipedia.org/wiki/Moleculehttps://en.wikipedia.org/wiki/Moleculehttps://en.wikipedia.org/wiki/DNAhttps://en.wikipedia.org/wiki/Molecular_biologyhttp://www.britannica.com/science/eukaryotehttp://www.britannica.com/science/endocytosishttp://www.britannica.com/science/adaptation-biology-and-physiologyhttp://www.britannica.com/science/evolution-scientific-theoryhttp://www.britannica.com/science/protozoanhttp://www.britannica.com/science/endocytosishttp://www.britannica.com/science/gonococcushttp://www.britannica.com/science/metabolismhttp://www.britannica.com/science/Haloarcula-marismortuihttp://www.britannica.com/science/Haloarcula-marismortuihttps://en.wikipedia.org/wiki/Moleculehttps://en.wikipedia.org/wiki/DNAhttps://en.wikipedia.org/wiki/Molecular_biology


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in clonin) or -hen s&clonin) inser's (NA in'o /ec'or (NA. All 'he 'erms can also &e

se in re+erence 'o $NA. The s'icky ens or cohesi/e ens +orm &ase pairs. Any '-o

complemen'ary cohesi/e ens can anneal, e/en 'hose +rom '-o i++eren' or)anisms.

This &ona)e is 'emporary, ho-e/er, an (NA li)ase -ill e/en'ally +orm a co/alen'

&on &e'-een 'he s)ar0phospha'e resie o+ a*acen' ncleo'ies 'o *oin 'he '-o

molecles 'o)e'her.

The simples' (NA en o+ a o&le s'rane molecle is calle a &ln' en. In a

&ln'0ene molecle &o'h s'rans 'ermina'e in a &ase pair . ln' ens are no' al-ays

esire in &io'echnolo)y since -hen sin) a (NA li)ase 'o *oin '-o molecles in'o one,

'he yiel is si)ni+ican'ly lo-er -i'h &ln' ens. hen per+ormin) s&clonin), i' also has

'he isa/an'a)e o+ po'en'ially inser'in) 'he inser' (NA in 'he opposi'e orien'a'ion

esire.

Non0&ln' ens are crea'e &y /arios o/erhan)s. An o/erhan) is a s're'ch o+

npaire ncleo'ies in 'he en o+ a (NA molecle. These npaire ncleo'ies can &e

in ei'her s'ran, crea'in) ei'her 37 or J7 o/erhan)s. These o/erhan)s are in mos' cases

palinromic.

The simples' case o+ an o/erhan) is a sin)le ncleo'ie. This is mos'

o+'en aenosine an is crea'e as a 37 o/erhan) &y some (NA polymerases. Mos'

commonly 'his is se in clonin) P"$ proc's crea'e &y sch an enyme. The

proc' is *oine -i'h a linear (NA molecle -i'h 37 'hymine o/erhan)s. Since aenine

an 'hymine +orm a &ase pair , 'his +acili'a'es 'he *oinin) o+ 'he '-o molecles &y a

li)ase, yielin) a circlar molecle.

VECTORS

A /ec'or is a (NA molecle 'ha' can &e se 'o inser' a (NA se1ence in'o a cell.Cec'ors are se +or replica'ion. =sal /ec'ors in la&ora'ories,

are plasmis , /irses 4@am&a Pha)e6, an ar'i+icial chromosomes. Cec'ors ms' &ecapa&le o+ &ein) replica'e &y 'he cell.

1. P@ASMI(0ASE( "@!NING CE"T!$S

• Plasmis are o&le s'rane, an commonly circlar. They are preispose

-i'h )ene'ic ma'erial, like 'he replica'ion ori)in 4-here (NA replica'ion s'ar's6 'o

https://en.wikipedia.org/wiki/Clone_(genetics)https://en.wikipedia.org/wiki/RNAhttps://en.wikipedia.org/wiki/Base_pairhttps://en.wikipedia.org/wiki/DNA_ligasehttps://en.wikipedia.org/wiki/DNA_ligasehttps://en.wikipedia.org/wiki/Nucleotidehttps://en.wikipedia.org/wiki/Adenosinehttps://en.wikipedia.org/wiki/DNA_polymerasehttps://en.wikipedia.org/wiki/PCRhttps://en.wikipedia.org/wiki/Thyminehttps://en.wikipedia.org/wiki/Base_pairhttp://en.wikipedia.org/wiki/Plasmidshttp://en.wikipedia.org/wiki/Viruseshttp://en.wikipedia.org/wiki/Plasmidhttps://en.wikipedia.org/wiki/Clone_(genetics)https://en.wikipedia.org/wiki/RNAhttps://en.wikipedia.org/wiki/Base_pairhttps://en.wikipedia.org/wiki/DNA_ligasehttps://en.wikipedia.org/wiki/Nucleotidehttps://en.wikipedia.org/wiki/Adenosinehttps://en.wikipedia.org/wiki/DNA_polymerasehttps://en.wikipedia.org/wiki/PCRhttps://en.wikipedia.org/wiki/Thyminehttps://en.wikipedia.org/wiki/Base_pairhttp://en.wikipedia.org/wiki/Plasmidshttp://en.wikipedia.org/wiki/Viruseshttp://en.wikipedia.org/wiki/Plasmid


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inepenen'ly replica'e insie o+ a &ac'erial hos' or yeas'. Al'ho)h plasmis

may ac' as parasi'es, 'hey are &ene+icial in -ays like an'i&io'ic resis'ance.

• !n one han, some plasmis are presen' in 1an'i'ies o+ one or '-o in a cell an

replica'e once per cell i/ision an are kno-n 'o &e ner s'rin)en' con'rol. !n

'he o'her han, mos' plasmis 'ilie in )ene'ic en)ineerin) are ner relae

con'rol -here as li''le as 9; an as many as ;; copies o+ plasmis can &e

presen' in a cell. In ai'ion, -hen pro'ein syn'hesis comes 'o a hal' e 'o

inhi&i'ion &y an an'i&io'ic, cell i/ision also s'ops an 'hese plasmis can

replica'e p-ars o+ 3,;;; copies in a cell. The 'ypes o+ plasmis syn'hesie

+rom )ene'ic en)ineerin) are ner relae con'rol, an con'ain )enes 'ha' are

resis'an' 'o an'i&io'ics, -hile carryin) enonclease si'es. These enonclease

si'es +acili'a'e 'he inser'ion o+ 'he (NA esire 'o &e copie. A ml'iple clonin)

si'e, or a polylinker is a small sec'ion o+ a (NA 'ha' has an array o+ res'ric'ion

si'es no' +on any-here else in 'he plasmi. p="98 4?plasmi =ni/ersal

"lonin)?6 is a commonly se /ec'or +rom E. coli.

2. CI$=S0ASE( "@!NING CE"T!$S

• Ano'her me'ho 'o clone (NA7s 'ha' are lar)er in sie is sin) a clonin) /ehicle

name &ac'eriopha)e lam&a. The cen'ral 'hir o+ 'he /irs7s )enome space can

&e inser'e &y (NA7s o+ lar)er sies &ecase 'ha' area is no' necessary rin)

pha)e in+ec'ion. In /i'ro me'hos can help 'he inser'ion o+ 'he chimeric pha)e

(NA &y in+ec'in) 'he hos' cells 'hro)h pha)es. ='ilia'ion o+ pha)es as clonin)

/ec'ors has a/an'a)es in 'ha' 'he chimeric (NA can &e mae in lar)e amon's

an easily pri+ie. In ai'ion, scien'is's can make se o+ lam&a pha)es +or lon)er (NA inser's. The only re1iremen' 'ha' is neee 'o allo- 'he /iral 'ool 'o

inser' (NA in'o 'he heas o+ pha)e is 'he 950&p se1ence kno-n as 'he cos si'e

a' each en. These ens nee 'o &e a' leas' 350J9 k& apar', an a com&ina'ion

o+ '-o cos si'es in /i'ro crea'es a cosmi /ec'or. "osmis o no' ha/e pha)e

)enes an 'here+ore can make plasmis.

• M930 +ilamen'os &ac'eriopha)e is an ano'her clonin) /ec'or 'ha' is sin)le0

s'rane an circlar (NA in a pro'ein '&e. The amon' o+ ien'ical helical

pro'ein s&ni's are epenen' on 'he len)'h o+ pha)e (NA &ein) coa'e, &' is

is normally Q2;; s&ni's. A lon)er ni' o+ pha)e molecle resl's -hen an

inser'ion o+ +orei)n (NA in a nonessen'ial re)ion occrs. The pha)es irec'lymake sin)le0s'rane (NA 'ha' a cer'ain 'echni1e re1ires, e/en 'ho)h M93

canno' main'ain (NA inser's lar)er 'han 9k&. This emons'ra'es a /isalia'ion

o+ -ha' an M93 looks like.

• Ano'her me'ho o+ /irs &ase clonin) is &aclo/irses, -hich are an array o+

lar)e an pa'ho)enic /irses 'ha' in+ec' insec's &' no' /er'e&ra'es, resl'in) in

an easy -ay 'o cl're 'hem in la&. @ike o'her /irses, a se)men' o+ 'he o&le

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s'rane (NA 'ha' +orms 'he )enome o+ 'he /irses is no' impor'an' +or /iral

replica'ion an can &e replace &y +orei)n (NA p-ar 'o 9Jk&.

3. %A" AN( A" CE"T!$

• In orer 'o accomoa'e (NA se)men's lar)er 'han 'hose carrie &y cosmis,yeas' ar'i+icial chromosomes 4%A"s6 an in &ac'erial ar'i+icial chromosomes

4A"s6. %A"s con'ain all moleclar necessi'ies o+ re1ire +or replica'ion +or

yeas' sch as 'he replica'ion ori)in, a'onomosly replica'in) se1ence 4A$S6,

a cen'romere. A"s replica'e in E. "oli, an are +on +rom plasmis 'ha'

normally replica'e lon) se)men's o+ (NA. A" /ec'ors ha/e 'he &are minimm

se1ences necessary +or sel+0replica'ion, copy nm&er con'rol, an re)la'e

plasmi separa'ion rin) cell i/ision.

GENETIC ENGINEERING

Gene'ic en)ineerin), also calle )ene'ic moi+ica'ion, is 'he irec' manipla'ion o+ an

or)anism7s )enome sin) &io'echnolo)y. I' is a se' o+ 'echnolo)ies se 'o chan)e 'he

)ene'ic makep o+ cells, inclin) 'he 'rans+er o+ )enes -i'hin an across species

&onaries 'o proce impro/e or no/el or)anisms. Ne- (NA may &e inser'e in 'he

hos' )enome &y +irs' isola'in) an copyin) 'he )ene'ic ma'erial o+ in'eres'

sin) moleclar clonin)me'hos 'o )enera'e a (NA se1ence, or &y syn'hesiin) 'he

(NA, an 'hen inser'in) 'his cons'rc' in'o 'he hos' or)anism. Genes may &e remo/e,or ?knocke o'?, sin) a nclease. Gene 'ar)e'in) is a i++eren' 'echni1e 'ha'

ses homolo)os recom&ina'ion 'o chan)e an eno)enos )ene, an can &e se 'o

ele'e a )ene, remo/e eons, a a )ene, or in'roce poin' m'a'ions.

An or)anism 'ha' is )enera'e 'hro)h )ene'ic en)ineerin) is consiere 'o &e

a )ene'ically moi+ie or)anism 4GM!6. The +irs' GM!s -ere &ac'eria)enera'e in

9>3 an GM mice in 9>. Inslin0procin) &ac'eria -ere commercialie in 9>82

an )ene'ically moi+ie +oo has &een sol since 9>>. GloFish, 'he +irs' GM!

esi)ne as a pe', -as +irs' sol in 'he =ni'e S'a'es in (ecem&er 2;;3. R9

Gene'ic en)ineerin) 'echni1es ha/e &een applie in nmeros +iels inclin)

research, a)ricl're, ins'rial &io'echnolo)y, an meicine. Enymes se in lanry

e'er)en' an meicines sch as inslin an hman )ro-'h hormone are no-

man+ac're in GM cells, eperimen'al GM cell lines an GM animals sch as mice

http://en.wikipedia.org/wiki/Centromerehttps://en.wikipedia.org/wiki/Genomehttps://en.wikipedia.org/wiki/Biotechnologyhttps://en.wikipedia.org/wiki/Organismshttps://en.wikipedia.org/wiki/DNAhttps://en.wikipedia.org/wiki/Molecular_cloninghttps://en.wikipedia.org/wiki/Geneshttps://en.wikipedia.org/wiki/Nucleasehttps://en.wikipedia.org/wiki/Gene_targetinghttps://en.wikipedia.org/wiki/Gene_targetinghttps://en.wikipedia.org/wiki/Homologous_recombinationhttps://en.wikipedia.org/wiki/Exonshttps://en.wikipedia.org/wiki/Point_mutationhttps://en.wikipedia.org/wiki/Genetically_modified_organismhttps://en.wikipedia.org/wiki/Bacteriahttps://en.wikipedia.org/wiki/Insulinhttps://en.wikipedia.org/wiki/Genetically_modified_foodhttps://en.wikipedia.org/wiki/GloFishhttps://en.wikipedia.org/wiki/Genetic_engineering#cite_note-1http://en.wikipedia.org/wiki/Centromerehttps://en.wikipedia.org/wiki/Genomehttps://en.wikipedia.org/wiki/Biotechnologyhttps://en.wikipedia.org/wiki/Organismshttps://en.wikipedia.org/wiki/DNAhttps://en.wikipedia.org/wiki/Molecular_cloninghttps://en.wikipedia.org/wiki/Geneshttps://en.wikipedia.org/wiki/Nucleasehttps://en.wikipedia.org/wiki/Gene_targetinghttps://en.wikipedia.org/wiki/Homologous_recombinationhttps://en.wikipedia.org/wiki/Exonshttps://en.wikipedia.org/wiki/Point_mutationhttps://en.wikipedia.org/wiki/Genetically_modified_organismhttps://en.wikipedia.org/wiki/Bacteriahttps://en.wikipedia.org/wiki/Insulinhttps://en.wikipedia.org/wiki/Genetically_modified_foodhttps://en.wikipedia.org/wiki/GloFishhttps://en.wikipedia.org/wiki/Genetic_engineering#cite_note-1


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or e&ra +ish are &ein) se +or research prposes, an )ene'ically moi+ie crops ha/e

&een commercialie.

•GENE CLONING

This escri&es 'he process o+ copyin) +ra)men's o+ (NA -hich can 'hen &e se +or

many i++eren' prposes, sch as crea'in) GM crops, or +inin) a cre +or isease.

There are '-o 'ypes o+ )ene clonin) in /i/o, -hich in/ol/es 'he se o+ res'ric'ion

enymes an li)ases sin) /ec'ors an clonin) 'he +ra)men's in'o hos' cells 4as can &e

seen in 'he ima)e a&o/e6. The o'her 'ype is in /i'ro -hich is sin) 'he polymerase

chain reac'ion 4P"$6 me'ho 'o crea'e copies o+ +ra)men's o+ (NA.

For in /i/o clonin) a +ra)men' o+ (NA, con'ainin) a sin)le )ene or a nm&er o+

)enes, is inser'e in'o a !"#$% 'ha' can &e ampli+ie -i'hin ano'her hos' cell. A /ec'or is

a sec'ion o+ (NA 'ha' can incorpora'e ano'her (NA +ra)men' -i'ho' losin) 'he capaci'y

+or sel+0replica'ion, an a /ec'or con'ainin) an ai'ional (NA +ra)men' is kno-n as

a &'(%)* !"#$% . I+ 'he +ra)men' o+ (NA incles one or more )enes 'he process is

re+erre 'o as +!,! "-$,),+.

POLYMERASE CHAIN REACTION

Polymerase chain reac'ion, 4P"$6, a 'echni1e se 'o make nmeros copies o+ a

speci+ic se)men' o+ (NA 1ickly an accra'ely. The polymerase chain reac'ion ena&les

in/es'i)a'ors 'o o&'ain 'he lar)e 1an'i'ies o+ (NA 'ha' are re1ire +or /arios

eperimen's an proceres inmoleclar &iolo)y, +orensic analysis,

e/ol'ionary &iolo)y, an meical ia)nos'ics.

P"$ -as e/elope in 9>83 &y :ary . Mllis, an American &iochemis' -ho -on 'he

No&el Prie +or "hemis'ry in 9>>3 +or his in/en'ion. e+ore 'he e/elopmen' o+ P"$, 'he

me'hos se 'o ampli+y, or )enera'e copies o+, recom&inan' (NA +ra)men's -ere 'ime0

consmin) an la&or0in'ensi/e. In con'ras', a machine esi)ne 'o carry o' P"$

reac'ions can comple'e many rons o+ replica'ion, procin) &illions o+ copies o+ a

(NA +ra)men', in only a +e- hors.

https://en.wikipedia.org/wiki/Zebrafishhttps://en.wikipedia.org/wiki/Genetically_modified_cropshttp://www.britannica.com/science/DNAhttp://www.britannica.com/science/DNAhttp://www.britannica.com/science/molecular-biologyhttp://www.britannica.com/science/biologyhttp://www.britannica.com/biography/Kary-B-Mullishttps://en.wikipedia.org/wiki/Zebrafishhttps://en.wikipedia.org/wiki/Genetically_modified_cropshttp://www.britannica.com/science/DNAhttp://www.britannica.com/science/DNAhttp://www.britannica.com/science/molecular-biologyhttp://www.britannica.com/science/biologyhttp://www.britannica.com/biography/Kary-B-Mullis


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The P"$ 'echni1e is &ase on 'he na'ral processes a cell ses 'o replica'e a ne-

(NA s'ran. !nly a +e- &iolo)ical in)reien's are neee +or P"$. The in'e)ral

componen' is 'he 'empla'e (NAi.e., 'he (NA 'ha' con'ains 'he re)ion 'o &e copie,

sch as a )ene. As li''le as one (NA molecle can ser/e as a 'empla'e. The only

in+orma'ion neee +or 'his +ra)men' 'o &e replica'e is 'he se1ence o+ '-o shor'

re)ions o+ ncleo'ies 4'he s&ni's o+ (NA6 a' ei'her en o+ 'he re)ion o+ in'eres'.

These '-o shor' 'empla'e se1ences ms' &e kno-n so 'ha' '-o primersshor'

s're'ches o+ ncleo'ies 'ha' correspon 'o 'he 'empla'e se1encescan &esyn'hesie. The primers &in, or anneal, 'o 'he 'empla'e a' 'heir complemen'ary si'es

an ser/e as 'he s'ar'in) poin' +or copyin). (NA syn'hesis a' one primer is irec'e

'o-ar 'he o'her, resl'in) in replica'ion o+ 'he esire in'er/enin) se1ence. Also

neee are +ree ncleo'ies se 'o &il 'he ne- (NA s'rans an a (NA polymerase,

an enyme 'ha' oes 'he &ilin) &y se1en'ially ain) on +ree ncleo'ies accorin)

'o 'he ins'rc'ions o+ 'he 'empla'e.

STEPS IN POLYMERASE CHAIN REACTION

9. (ena'ra'ion or separa'ion o+ 'he '-o s'rans o+ 'he (NA molecle. This is

accomplishe &y hea'in) 'he s'ar'in) ma'erial 'o 'empera'res o+ a&o' >J "

42;3 F6. Each s'ran is a 'empla'e on -hich a ne- s'ran is &il'.

2. In 'he secon s'ep 'he 'empera're is rece 'o a&o' JJ " 4939 F6 so 'ha' 'he

primers can anneal 'o 'he 'empla'e. In 'he 'hir s'ep 'he 'empera're is raise 'o

a&o' 2 " 4952 F6, an 'he (NA polymerase &e)ins ain) ncleo'ies on'o

'he ens o+ 'he anneale primers.

http://www.britannica.com/science/template-replicationhttp://www.britannica.com/science/genehttp://www.britannica.com/science/nucleotidehttp://www.britannica.com/science/oligonucleotidehttp://www.britannica.com/science/enzymehttp://www.britannica.com/science/template-replicationhttp://www.britannica.com/science/genehttp://www.britannica.com/science/nucleotidehttp://www.britannica.com/science/oligonucleotidehttp://www.britannica.com/science/enzyme


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3. A' 'he en o+ 'he cycle, -hich las's a&o' +i/e min'es, 'he 'empera're is raise

an 'he process &e)ins a)ain. The nm&er o+ copies o&les a+'er each cycle.

=sally 2J 'o 3; cycles proce a s++icien' amon' o+ (NA.

COMPONENTS OF POLYMERASE CHAIN REACTION

The P"$ reac'ion re1ires 'he +ollo-in) componen's

9. (NA TEMP@ATE

• The sample (NA 'ha' con'ains 'he 'ar)e' se1ence. A' 'he &e)innin) o+ 'he

reac'ion, hi)h 'empera're is applie 'o 'he ori)inal o&le0s'rane (NA

molecle 'o separa'e 'he s'rans +rom each o'her.

2. (NA P!@%ME$ASE

• A 'ype o+ enyme 'ha' syn'hesies ne- s'rans o+ (NA complemen'ary 'o 'he

'ar)e' se1ence. The +irs' an mos' commonly se o+ 'hese enymes

is Taq (NA polymerase 4+romThermis aquaticus6, -hereas Pfu (NA polymerase

4+romPyrococcus furiosus6 is se -iely &ecase o+ i's hi)her +ieli'y -hen

copyin) (NA. Al'ho)h 'hese enymes are s&'ly i++eren', 'hey &o'h ha/e '-o

capa&ili'ies 'ha' make 'hem si'a&le +or P"$ 96 'hey can )enera'e ne- s'rans

o+ (NA sin) a (NA 'empla'e an primers, an 26 'hey are hea' resis'an'.

3. P$IME$S

• Shor' pieces o+ sin)le0s'rane (NA 'ha' are complemen'ary 'o 'he 'ar)e'

se1ence. The polymerase &e)ins syn'hesiin) ne- (NA +rom 'he en o+ 'he

primer.

. N="@E!TI(ES

• Sin)le ni's o+ 'he &ases A, T, G, an ", -hich are essen'ially ?&ilin) &locks?

+or ne- (NA s'rans.

INSTRUMENTS USED IN POLYMERASE CHAIN REACTION

• A 'hermocycler or P"$ machine is a la&ora'ory appara's se +or P"$. The

e/ice has a 'hermal &lock -i'h holes -here '&es -i'h 'he P"$ reac'ion

mi'res can &e inser'e. The cycler 'hen rises an lo-ers 'he 'empera're o+ 'he


13/13

&lock in iscre'e, pre0pro)ramme s'eps. Thermal cyclers are e1ippe -i'h ho'

&onne', -hich is a hea'e pla'e 'ha' presses a)ains' 'he lis o+ 'he reac'ion

'&es. This pre/en's conensa'ion o+ -a'er +rom 'he reac'ion mi'res 'o 'he

insies o+ 'he lis an makes i' nnecessary 'o se P"$ oil

APPLICATIONS OF POLYMERASE CHAIN REACTION

9. Pri+ica'ion o+ ncleic acis +rom en/ironmen'al microor)anisms +or P$"

ampli+ica'ion2. Pri+ica'ion o+ ncleic aci +rom soil an seimen'3. Pri+ica'ion in ncleic acis +rom microor)anisms in -a'er . To eplore /arios areas en/ironmen'al micro&iolo)y has 'he po'en'ial 'o sol/e

many i++icl' an nans-ere 1es'ions a&o' micro&ial ac'i/i'ies in 'he

en/ironmen' a' 'he physiolo)ical an moleclar s'a)e.J. For 'he e'ec'ion o+ speci+ic micro&es in en/ironmen'al samples

biochemistry finals

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