Improvement of PCR primer design for detection of prokaryotic species
Date
2012-06-22
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Abstract
Polümeraasi ahelreaktsioon ehk PCR on molekulaarbioloogia meetod, mis võimaldab paljundada spetsiifilist DNA lõiku. Protsess toimub tsükliliselt ja kätkeb endas järgmisi etappe: kaheahelalise DNA järjestuse (sihtmärkjärjestuse) sulatamist kõrgel temperatuuril, kahe spetsiifilise DNA järjestuse (PCRi praimeri) seondumist sihtmärkjärjestusele konkreetsest PCRi katsest sõltuval madalamal temperatuuril (ehk praimerite sulamistemperatuuril Tm) ning seondunud praimerite pikendamist vastavalt spetsiifilisele DNA lõigu järjestusele kindla valgulise ensüümi abil. Paljundatud DNA-d detekteeritakse, kas spetsiifiliselt geelil pikkuse järgi või reaalajas produkti paljundamise käigus tekkiva signaali abil. PCR võimaldab sel viisil tuvastada suvalisest DNA proovist (kliiniline-, veterinaar-, toidu-, keskkonnaproov jne) vaid kindlale liigile iseäralikku DNA järjestust ning seetõttu on tehnoloogia leidnud rakendust erinevates valdkondades erinevate liikide või tüvede tuvastamiseks.
Üks olulisemaid eeldusi edukaks PCRi teostamiseks on täpsete ja tundlikke praimerite disainimine (PCRi praimeridisain). PCRi praimeridisain sisaldab endas erinevaid etappe muuhulgas sihtmärkjärjestuse välja valimist (nt kindla järjestuse valimist bakterigenoomist) ning PCRi praimerijärjestuste disaini.
Käesolev töö ongi keskendunud PCRi praimeridisaini erinevate etappide parendamisele: laialdaselt kasutusel oleva PCRi praimeridisaini programmi Primer3-e poolt kasutatava praimerite sulamistemperatuuri Tm arvutamise valemi täiustamisele ning sellele, kuidas prokarüootseid liigispetsiifilisi kordusjärjestusi PCRi sihtmärkjärjestustena kasutada ning, millise effekti see PCRi tulemustele annab. Viimase osana sisaldab töö prokarüootsete liigispetsiifiliste kordusjärjestuste iseloomustamist 613 erinevas prokarüootses liigis.
Antud töö tulemused aitavad kaasa uute ja paremate molekulaardiagnostika testide loomisele esiteks lihtsustades nende väljatöötamist tänu antud töös leitud ja kirjeldatud PCRi sihtmärkjärjestustele, teiseks tõstes nende töökindlust, kuna liigispetsiifilistele kordusjärjestustele disainitud praimerid on kõrgendatud tundlikkusega ning, kuna parendatud praimeridisaini-programm Primer3 võimaldab disainida praimereid, mis vastavad täpsemalt etteantud molekulaardiagnostika testide tingimustele .
Polymerase chain reaction or PCR is a method in molecular biology, which enables amplification of specific DNA regions. It is a cyclic process, that comprises of heat denaturation of double-stranded DNA (target sequence), hybridization of two short oligonucleotides (called PCR primers) to the denaturated target sequence at temperature specific to the PCR reaction (termed melting temperature Tm) and extension of hybridized primers by the enzyme DNA polymerase. The amplified DNA can be identified either specifically on electrophoresis gel by its product length or by monitoring the signal generated through activation of primer attached flourecent labels during the amplification in real-time. Thus, PCR enables identification of the DNA sequence of interest from an arbitrary DNA sample (e.g. clinical, veterinary, food, environmental sample) and therefore it is widely applied in different areas for particular species or strain identification. The design of specific and sensitive PCR primers is very important for the success of PCR. PCR primer design comprises of different steps, among which choice of the target sequence (e.g. certain DNA region in a bacterial genome) and the design of the primer sequences are crucial. Current thesis is focused on the improvement of the different steps of PCR primer design. First, enhancements to widely used PCR primer design program Primer3 are introduced. These enhancements enable to calculate primer melting temperature more accurately (includes modernization of the primer melting temperature formula as well as the auxiliary formulas that enable to take the concentrations of salt ions in the PCR reaction buffer into consideration). Second, we provide a methodology for finding the prokaryotic species-specific repeats and their application as PCR targets in primer design. Also, the positive impact to PCR sensitivity of inclusion of repetitive sequences as PCR targets is ascertained. The last part of this thesis covers the characterization of species-specific repetitive sequences in 613 different prokaryotic species. The results of current thesis facilitate the design of new and more reliable tests to molecular diagnostics because of the followings: first, new PCR target sequences are introduced, second, we have shown that primers designed to species-specific repetitive sequences increase the sensitivity of PCR and third enhanced primer design program enables to design primers that follow more precisely the preset conditions of tests in molecular diagnostics.
Polymerase chain reaction or PCR is a method in molecular biology, which enables amplification of specific DNA regions. It is a cyclic process, that comprises of heat denaturation of double-stranded DNA (target sequence), hybridization of two short oligonucleotides (called PCR primers) to the denaturated target sequence at temperature specific to the PCR reaction (termed melting temperature Tm) and extension of hybridized primers by the enzyme DNA polymerase. The amplified DNA can be identified either specifically on electrophoresis gel by its product length or by monitoring the signal generated through activation of primer attached flourecent labels during the amplification in real-time. Thus, PCR enables identification of the DNA sequence of interest from an arbitrary DNA sample (e.g. clinical, veterinary, food, environmental sample) and therefore it is widely applied in different areas for particular species or strain identification. The design of specific and sensitive PCR primers is very important for the success of PCR. PCR primer design comprises of different steps, among which choice of the target sequence (e.g. certain DNA region in a bacterial genome) and the design of the primer sequences are crucial. Current thesis is focused on the improvement of the different steps of PCR primer design. First, enhancements to widely used PCR primer design program Primer3 are introduced. These enhancements enable to calculate primer melting temperature more accurately (includes modernization of the primer melting temperature formula as well as the auxiliary formulas that enable to take the concentrations of salt ions in the PCR reaction buffer into consideration). Second, we provide a methodology for finding the prokaryotic species-specific repeats and their application as PCR targets in primer design. Also, the positive impact to PCR sensitivity of inclusion of repetitive sequences as PCR targets is ascertained. The last part of this thesis covers the characterization of species-specific repetitive sequences in 613 different prokaryotic species. The results of current thesis facilitate the design of new and more reliable tests to molecular diagnostics because of the followings: first, new PCR target sequences are introduced, second, we have shown that primers designed to species-specific repetitive sequences increase the sensitivity of PCR and third enhanced primer design program enables to design primers that follow more precisely the preset conditions of tests in molecular diagnostics.
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Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
polümeraasahelreaktsioon, molekulaardiagnostika, polymerase chain reaction, molecular diagnostics