Teknikat molekulare t ё k ё rkimit

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Teknikat mё tё pёrdorura nё nivel molekular qё pёrdoren nё kёrkime tё bioteknologjisё molekulare e tё aplikuar:

Sinteza kimike e ADN-sё Teknikat e pёrcaktimit tё renditjes sё

nukleotideve tё ADN-sё (sekuencimit) Teknika e PCR-sё (Polymerase Chain Reaction) Metoda e fitimit tё antitrupave monoklonalё si

dhe pёrdorimet e tyre

2011/2012teknikat molekulare tё kёrkimit

Teknikat molekulare tё kёrkimit

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Zinxhirёt polinukleotidikё, qё sintetizohen mund tё pёrdoren pёr qёllime tё shumta:

Pёr tё formuar gjene tё plota apo fragmente tё tyre Pёr tё amplifikuar fragmente tё caktuara ADN-je Pёr tё shkaktuar mutacione tё ADN- ja e izoluar

paraprakisht Si sonda pёr hibridizimnin e ADN-sё Ku kryhet sinteza kimike e oligonukleotideve?- Nё aparatura tё kompjuterizuara SINTETIZATORE automatik

tё ADN-sё- E pёrbёrё nga njё sёrё pompash dhe valvulash qё

programohen pёr tё futur nё mёnyrё precize tё gjithё reaktivёt kimik dhe nukleotidet qё zgjasin zinxhirin oligoinukleotidik.


Sinteza kimike e ADN-sё - in vitro

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Sinteza in vitro kryhet nё drejtimin 3’-5’, cdo hap dhe kohёzgjatja kontrollohen nё mёnyrё automatike .

Metoda mё e njohur pёr sintetizimin kimik ёshtё ajo e fosforamiditit

Nucleoside phosphoramidites are derivatives of natural or synthetic nucleosides. They are used to synthesise short nucleic acid chains.

Nucleosides are glycosylamines consisting of a nucleobase (often referred to as simply base) bound to a ribose or deoxyribose sugar via a beta-glycosidic linkage. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine.


Sinteza kimike e ADN-sё

http://en.wikipedia.org/wiki/Nucleoside

http://en.wikipedia.org/wiki/Oligonucleotide_synthesis

http://en.wikipedia.org/wiki/Nucleic_acid

http://en.wikipedia.org/wiki/Glycosylamine

http://en.wikipedia.org/wiki/Nucleobase

http://en.wikipedia.org/wiki/Ribose

http://en.wikipedia.org/wiki/Deoxyribose

http://en.wikipedia.org/wiki/Sugar

http://en.wikipedia.org/wiki/Anomer

http://en.wikipedia.org/wiki/Glycosidic_bond

http://en.wikipedia.org/wiki/Glycosidic_bond

http://en.wikipedia.org/wiki/Cytidine

http://en.wikipedia.org/wiki/Uridine

http://en.wikipedia.org/wiki/Adenosine

http://en.wikipedia.org/wiki/Guanosine

http://en.wikipedia.org/wiki/Thymidine

http://en.wikipedia.org/wiki/Inosine



SINTETIZATORI OSE SINTIZAJZER

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Para fillimit tё reaksionit tё gjitha nukleozidet trajtohen duke zёvendёsuar aminogrupet e bazave tё tyre me grupe benzoile , qё mos tё vije nё reaksione tё padёshiruara

Nukleozidi i parё qё do pёrbёjё skajin 3’ tё zinxhirit sintetizues fillimisht lidhet me njё suport tё ngurtё (sfera xhami) (fiksim), me ndёrmjetёsin e njё grupi qё quhet spejser.

Ndёrsa nё skajin 5’ tё kёtij nukleozidi lidhet njё grup DMT (DIMETOKSITRITIL)., pёr tё mos lejuar qё ky (nukleozidi) tё hyjё nё lidhje. Ky komponim quhet edhe komleksi nismёtar i sintezёs kimike.



http://upload.wikimedia.org/wikipedia/commons/6/6d/Benzoyl_group.png


Adenozina → benzoil group

http://upload.wikimedia.org/wikipedia/commons/6/6d/Benzoyl_group.png

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Cdo nukleotid i ri qё do i shtohet kompleksit fillestar modifikohet duke lidhur nё skajin 5’ njё grup DMT ndёrsa nё atё 3’ njё grup diizopropilamin (kripe litiumi).

Diisopropylamine is a secondary amine with the chemical formula (CH3)2HC-NH-CH(CH3)2

Kjo strukturё e nukleotideve qё shtohen nё reaksion quhet nukleosid FOSFORAMIDIT, nga emri I metodёs.

Procesi I zgjatjes bazohet nё alternimin e largimit tё DMT DIMETOKSITRITIL, duke lёnё tё lirё skajin 5’ dhe shtimin e nukleozideve tё reja nё formёn e fosforamiditit.

Nga reaksione tё njёpasnjёshme tё kёtij lloji formohet zinxhiri i plotё i kёrkuar sipas sekuencёs sё kёrkuar paraprakisht



http://en.wikipedia.org/wiki/Secondary_amine

http://en.wikipedia.org/wiki/Chemical_formula


The 5'-hydroxyl group is protected by an acid-labile DMT (dimethoxytrityl) group.

Bz (benzoyl) protection is used for A, dA, C, and dC, and G and dG are protected ёith isobutyryl groupAc (acetyl) group is often used to protect C and dC as shoёn in Figure

cytidine, adenosine, guanosine, thymidine

http://en.wikipedia.org/wiki/Protecting_group#Common_protecting_groups.23Alcohol_protecting_groups

http://en.wikipedia.org/wiki/Protecting_group#Common_protecting_groups.23Amine_protecting_groups

http://en.wikipedia.org/wiki/Protecting_group#Common_protecting_groups.23Amine_protecting_groups

http://en.wikipedia.org/wiki/Adenosine

http://en.wikipedia.org/wiki/Guanosine

http://en.wikipedia.org/wiki/Thymidine

http://upload.wikimedia.org/wikipedia/commons/d/dd/Phosphoramidite1.png


Metoda e FOSFORAMIDIT

fosforamiditi

nukleozid

Largimi i diizopropilaminit nga fosforamiditiOrientimi 3’ 5’

Largimi I DMT

http://upload.wikimedia.org/wikipedia/commons/3/38/Oligocycle1.png

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Reaksioni caping dhe oksidimit – nёnkupton largimin e grupeve metile qё janё tё lidhura nё grupin fosfat, largimi I grupeve mbrojtёse tё grupit aminik dhe largimi I DMT-sё

Pas kёtyre reaksioneve ologonukleotidet purifikohen dhe pёrdoren pёr qёllime e paracaktuara.

APLIKIMI I OLIGONUKLEOTIDEVE Nё BIOTEKNOLOGJI



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Ologonukleotidet (having feё, having little) qё sintetizohen nё mёnyrё sintetike pёrdoren si:

Sonda pёr eksperimentimin e hibridizimit tё ADN-sё

Si sekuenca prajmere pёr sekuencimin e ADN-sё

Pёr tё ndryshuar renditjen e gjeneve tё klonuara

Small interfering RNA


APLIKIMI/RёNDёSIA E OLIGONUKLEOTIDEVE Nё BIOTEKNOLOGJI


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Oligonukleotide me zinxhirё tё njёfishtё prej 40-60 bazash, mund tё sintetizohen bazuar nё renditjen e njё aminoacidi

Zinxhirё tё dyfishtё tё ADN-sё linker, (sekuence palindromike qё mund tё mbyllet nё vetevete, me pozicione restriktive, mund tё pёrdoren si lidhёs me vektorё

Oligomere tё pёrbёrёr prej 17-24 nukleotidesh qё mund tё pёrdoren si prajmerё pёr reaksionet e sekuencimit tё ADN

Oligomerё me zinxhirё tё njwfishtё mund tё pёrdoren pёr mutagjenzёn “in vitro”


Produktet e sintezёs kimike tё ADN-sё ?????


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Sinteza e gjeneve nga 60-80 cb, fillimisht vargjet komplementare sintetizohen nё formёn e segmenteve pёrbёrёse prej njё zinxhiri por qё janё tё mbivendoshёm njёri mbi tjerin, ndёrhyhet me T4 ligazё

Gjenet mё tё mёdha nga 300 cb, kanё gabime gjatё sintetizimit in vitro, pёrdoret njё strategji tjetёr: edhe kёto formohen zinxhirёt e gjatё tё njwfishtё POR FRAGMENTE JOTёPLOTA, kur komlementohen me njёri tjetrin ngelin vende qё nuk mbulojnё tё gjithё gjatёsine e dy vargjeve, NDёRHYHET ME ADN POLIMERAZё-I, qё “mbushё” hapёsirat e njefishta, ndёrsa T4 ligaza, i lidhё zonat e kёputura, ky proces quhet asemblim in vitro


Sinteza e gjeneve


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The term DNA sequencing refers to sequencing methods for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA.

Shfrytёzohet nё: biotechnology, forensic biology and biological systematics

Teknika mё e njohur ёshtё Maxam dhe Gilbert (1976) dhe metoda sipas Sanger

Sipas MG, zinxhirit tё ADN-sё qё kёrkohet renditja shёnohet nё mёnyrё radioaktive nё skajin 5’, mё pas ADN-ja trajtohet kimisht nё mёnyrё qё tё prishen lidhjet midis bazave.


Teknikat e sekuencimit tё ADN-sё

http://en.wikipedia.org/wiki/Sequencing

http://en.wikipedia.org/wiki/Nucleotide

http://en.wikipedia.org/wiki/Adenine

http://en.wikipedia.org/wiki/Guanine

http://en.wikipedia.org/wiki/Cytosine

http://en.wikipedia.org/wiki/Thymine

http://en.wikipedia.org/wiki/DNA

http://en.wikipedia.org/wiki/Biotechnology






http://en.wikipedia.org/wiki/Forensic_biology

http://en.wikipedia.org/wiki/Systematics


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Krijohen kushte tё tilla qё nё katёr reaksione tё ndryshme pёr, A,G, C,T tё nxitet prerja nё njёrёn nga bazat.

Pas elektroforezёs nё 4 shirita, vec pёr cdo reaksion dhe pas autoradiografisё , mund tё pёrcaktohet renditja e bazave tё zinxhirit tё analizuar nё drejtimin 3’-5’


Teknikat e sekuencimit tё ADN-sё


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Because the chain-terminator method (or Sanger method after its developer Frederick Sanger) is more efficient and uses feёer toxic chemicals and loёer amounts of radioactivity than the method of Maxam and Gilbert, it rapidly became the method of choice. The key principle of the Sanger method ёas the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators

Dideoksinukleotidet janё nukleotide tё sintetizura nё rrugё kimike ku mungojnё karbonet 2’ dhe 3’ tё pentozёs


chain-terminator method, Sanger

http://en.wikipedia.org/wiki/Frederick_Sanger

http://en.wikipedia.org/wiki/Dideoxynucleotides



The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation.

The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase.

To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) ёhich are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond betёeen tёo nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length.

http://en.wikipedia.org/wiki/Primer_(molecular_biology)

http://en.wikipedia.org/wiki/DNA_polymerase

http://en.wikipedia.org/wiki/Fluorescence

http://en.wikipedia.org/wiki/Deoxynucleotides

http://en.wikipedia.org/wiki/DNA_polymerase

http://en.wikipedia.org/wiki/Dideoxynucleotides

http://en.wikipedia.org/wiki/Hydroxyl

http://en.wikipedia.org/wiki/Phosphodiester_bond

http://en.wikipedia.org/wiki/Phosphodiester_bond

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• The neёly synthesized and labeled DNA fragments are heat denatured, and separated by size (ёith a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel ёith each of the four reactions run in one of four individual lanes (lanes A, T, G, C); • the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. • In the image on the right, X-ray film ёas exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence 2011/2012teknikat molekulare tё kёrkimit

http://en.wikipedia.org/wiki/DNA_denaturation

http://en.wikipedia.org/wiki/Gel_electrophoresis

http://en.wikipedia.org/wiki/Polyacrylamide_gel

http://en.wikipedia.org/wiki/Autoradiography

http://en.wikipedia.org/wiki/Radiography


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http://en.wikipedia.org/wiki/File:Sequencing.jpg


Sinteza kimike e AND-sёDNA SEKUENCATORI

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Pra sinteza do tё ndalet nё nivelin e cdo baze tё atij lloji,

Pra pritet qё p.sh nё tubin e reaksionit pёr adeninёn do formohen zinxhirё me gjatёsi progresive sinteza e tё cilёve ndalet nё cdo bazё adeninё qё ajo has.

Ndahet zinxhiri I dyfishte me FORMAMID, dhe mё pas nё zhel polakrilamid (aftesi tё lartё fraksionuese dhjetra baza), dhe bandat bёhen tё dukshme ose me antё tё radiografisё ose me anё tё ngjyrosjes specifike.

Sot, nё metodёn Sanger shfrytёzohet shenues fluoreshent nё vend radioaktivё, pёr cdo bazё. Kjo ka bёrё tё mundur automatizimin e kёsaj metode.


Metoda e dideoksinukleotideve(Sanger)

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Dideoksinukleotidet janё nukleotide tё sintetizura nё rrugё kimike ku mungpjnё karbonet 2’ dhe 3’ tё pentozёs

Dezoksiribonukleotidet natyrale grupin hidroksil e kanё nё pozcionin 3’ (kujto 5’-3’), pra grupi 3’OH ёshtё grupi reaktiv qё mer pjesё nё lidhjen midis nukleotideve nё sintesёn normale tё AND-sё in vivo. Kjo do tё thotё se nёsё inkoroporohet njё dideoksinukleotid , zinxhiri nuk ёshtё mё nё gjёndje tё zgjatet.

Nё kёtё metodё fillimisht ndahen zinxhirёt, njёri nga vargjet I nёnshtrohet reaksioneve tё sekuencimit.

Fillimisht, nё zinxhirin e reaksioneve njёrit varg (zinxhirit ta njfishtё) tё ADN-sё I shtohet njё oligonukleotid qё do tё shёrbejё si prajmer pёr tё vazhduar sintetizimin.


Metoda e dideoksinukleotideve(Sanger)

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Nё cdo tub reaksioni shtohen tё gjitha dezoksirbonukleotidtrifosfatet (dNTP), dATP, dTTP,dCTP, dhe dGTP, ku vetёm njёri ёshtё I shёnuar nё secilin prej reaksioneve me lёndё radioaktive ose joradioaktive, dhe nё secilin tub ne vecanti shtohet nga njёri prej dideoksiribonukleotidtrifosfateve ddATP, ddTTP, ddGTP, ddCTP, nё pёrqёndrime tё tilla qё tё mund tё inkorporohen nё zinxhirin nё rritje nё tё gjitha vendet e mundshme pёr to.

Pas reaksioneve nё secilin prej tubave nё prani tё ADN-polimerazёs, do tё ndodhё procesi i zgjatjes sё zinxhirёve tё rinj deri sa reaksioniinkorporon nje dd.

PёRPARёSI: Sekuencohet edhe ARN, revesrz transkriptaza pёr tё kthyer ne cDNA komplementare., mё pas behet sekuencimi si mё lartё.


Metoda e dideoksinukleotidevechain termination (Sanger)

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Me metodёn: Pёrdorimi I fagut M13, sekuencohen deri nё 500

cb, Dhe primer ёalking, disa mijёra CB , prajmer qё

ecin, sintetizohen prajmer qё drejtojnё sintezёn nё 200-250 nukleotide.


Sekuencimi I fragementeve mё tё mёdha

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Metoda e reaksionit zinxhirё tё polimerazёs, pёrdoret pёr amplifikimin (shumimin) e fragmenteve tё interesit tё ADN-sё, deri nё miliona kopje in vitro .

Zbuluesi i kёsaj metode Kary Mullis, fitues i çmimit Nobёl me dr. Micheal Smith, nё kiminё e acideve nukleike.

Parakushti kryesor pёr shumimin e fragmentit tё ADN-sё ёshtё njohja e sё paku njё pjese tё vogёl tё sekuencёs nukleotide qё mundёson sintezёn e sekuencave oligonukleotideve komplementare njёzinxhirore, pra sic njihen ndryshe “primer” me 3OH fund.


Reaksioni zinxhir i polimerazёsPCR – polymerase chain reaction

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Çfar duhet pёr reaksionin fillestar tё PCR-sё?

Molekulё e ADN-sё me njё gjen ose sekuencёn target tё cilёn duam tё amplifikojmё

Tё katёr deoksiribonukleotidtrifosfatet (dATP, dGTP, dCTP, dTTP)

Sekuencat fillestare tё oligonukleotideve (prajmerёt)

dhe ADN polimeraza (zakonisht Taq polimeraza)



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Njё pёrzierje e pёr PCR nё 25 uL konsiston nё:

1. QH2O 15,5 μL2. Master mix 8,0 μL (5 x puffer TaqMaster, 10

pufer, dNTP (10mM)3. Primer nga secili 0,5 μL4. ADN nga 0.5 μL5. Polimerza Taq 0.5 μL


Reaksioni zinxhir i polimerazёsPCR – polymerase chain reactionpёrvoja nga puna e magjistraturёs

http://en.wikipedia.org/wiki/File:PCR_tubes.png



GeneAmp PCR System 2400 (Applied Biosysytems)


Reaksioni zinxhir i polimerazёssi duket njё program i PCR-sё ?

t° denaturimit

t° e hibridizimit

t° e elongacionit

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Pёr detektimin e sasive tё vogla tё AND-sё nё kampione si floku, sasi shumё tё vogla gjaku, kocka shumё tё vjetra, pjesё indesh tё degjeneruara, nё diagnostifikimin e sёmundjeve gjentike, identifikimin e mutacioneve, detektimi I baktereve, viruseve, nё industrinё ushёimore, sondave molekulare , revolucion nё bilogjinё eksperimentale

These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.


Pёrdorimi i PCR-sё

http://en.wikipedia.org/wiki/DNA_cloning

http://en.wikipedia.org/wiki/DNA_sequencing

http://en.wikipedia.org/wiki/Phylogeny

http://en.wikipedia.org/wiki/Gene

http://en.wikipedia.org/wiki/Hereditary_disease

http://en.wikipedia.org/wiki/Genetic_fingerprint

http://en.wikipedia.org/wiki/Forensic_science

http://en.wikipedia.org/wiki/Parental_testing

http://en.wikipedia.org/wiki/Infectious_disease

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Second part next week


Second part next week,

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Monoclonal antibodies (mAb or moAb) are monospecific antibodies (proteina specifike) that are the same because they are made by identical immune cells that are all clones of a unique parent cell.

Si formohen nё rrugё in vitro: qelizat e mielomёs sё miut (tumorale qё shumohen nё mёnyrё tё pakontrolluar, por nuk prodhojnё antitrupa) u shkrinё me qeliza normale tё shpretkёs tё marra nga njё mi i imunizuar ndaj njё antigjeni tё dhёnё. Kёto tё fundit prodhojnё antitrupat specifik ndaj antigjenit specifik, por nuk riten shpejt.

Nga fuzionimi e kёtyre qelizave u fituan qeliza hibride somatike (HIBRIDOMAT) ku ishin tё kombinuara cilёsitё pozitive tё tё dyja qelizave.


Antitrupat monoklonalё

http://en.wikipedia.org/wiki/Monospecific_antibody

http://en.wikipedia.org/wiki/Monospecific_antibody

http://en.wikipedia.org/wiki/White_blood_cell

http://en.wikipedia.org/wiki/Cloning


Formimi i hibridomave dhe antitrupave monoklonaledhe pёrzgjedhja me HGPRT-hipoksantine-guaninё-fosforibozil-transferaza)

(hipoksantinё-aminopterine-timidinё)

http://upload.wikimedia.org/wikipedia/commons/9/9a/Monoclonals.png

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Detektimi i antitrupave tё cliruar nga qelizat prodhuese

Lidhja e antitrupit me antigjenin me tё cilin ёshtё inokuluar kafsha nga janё nxjerrё qelizat fillestare,

Lidhja antigjen-antitrup ёshtё e qёndrueshme dhe pas lidhjes me njё antitrup sekondar vihet nё dukje prania e antitrupit tё deshiruar. Antitrupi sekondar lidhet mё njё enzimё, qё shndron njё substrat pa ngjyrё nё substrat tё ngjyrosur. Pra klonet qё shfaqin substrta tё ngjyrosur janё linjat hibride antitrup prodhuese tё dёshiruara.


Si identifikohen hibridёt e dёshiruar


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1. Pёrdoren pёr ndarjen e interferoneve (Interferons (IFNs) are proteins made and released by lymphocytes in response to the presence of pathogens—such as viruses, bacteria, or parasites—or tumor cells) nga qelizat.

Antitrupi monoklonal lidhet pёr interferonin specifik, ne kromatografine nёkollonё. (bёhet shpёrlarja e interferonit nga antitrupi, pastrim 5000 herё mё I lartё se cdo rrugё tjetёr.

2. Pёr tipizimin qelizor – AM pёrbёjnё mjete shumё specifike pёr identifikimin e antigjeneve specifikё tumoralё nё melanomat, karcinomat, etj.


Pёrdorimi i antitrupave monoklonalё

http://en.wikipedia.org/wiki/Protein

http://en.wikipedia.org/wiki/Lymphocyte

http://en.wikipedia.org/wiki/Pathogen

http://en.wikipedia.org/wiki/Virus

http://en.wikipedia.org/wiki/Bacteria

http://en.wikipedia.org/wiki/Parasite

http://en.wikipedia.org/wiki/Tumor


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3. Nё diagnostikё dhe terapi, - identifikimi i viruseve ose baktereve nё nja material biologjik nёse ёshtё I disponueshёm antitrupi specifik ndaj tij.

Lidhja e antitrupave monoklonalё me antigjenen specifike tё disa tumoreve humane , si p.sh atё tё antimielomёs etj.


Pёrdorimi i antitrupave monoklonalё


Teknikat molekulare t ё k ё rkimit

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