GDNF geeni sekveneerimine ja mutatsioonide analüüs ...

SisukordSisukord ...................................................................................................................................... 2Kasutatud lühendid ..................................................................................................................... 3Sissejuhatus ................................................................................................................................ 41. Kirjanduse ülevaade .......................................................................................................... 51.1 Neerude ja kuseteede areng .......................................................................................... 51.2 Kaasasündinud neeru ja kuseteede anomaaliad ............................................................ 71.3 GDNF geen ja valk ....................................................................................................... 81.3.1 GDNF/RET signaalrada ...................................................................................... 91.4 DNA varieeruvus ........................................................................................................ 101.4.1 Kodeeriva ala mutatsioonid .............................................................................. 101.4.2 Regulatoorse ala mutatsioonid .......................................................................... 111.4.3 Mutatsioonide mõju fenotüübile ....................................................................... 111.5 DNA sekveneerimine ................................................................................................. 121.5.1 Sangeri meetod .................................................................................................. 121.5.2 Teise põlvkonna sekveneerimine ...................................................................... 131.5.2.1 Illumina/Solexa ........................................................................................ 141.5.2.2 454 pürosekveneerimine .......................................................................... 151.5.2.3 ABi SOLiD .............................................................................................. 151.5.2.4 Teise generatsiooni sekveneerimistehnoloogiate eelised ........................ 161.6 SNP-de genotüpiseerimine ......................................................................................... 171.6.1 Taqmani meetod SNP-de genotüpiseerimiseks ................................................. 171.6.2 PCR-i ja geelipõhised meetodid ........................................................................ 181.6.2.1 Alleelspetsiifiline PCR ............................................................................ 181.6.2.2 PCR-RFLP ............................................................................................... 191.6.3 PCR-i vabad genotüpiseerimismeetodid ........................................................... 201.6.4 SNP-de analüüs Illumina Infinium® tehnoloogiaga ......................................... 201.7 Genotüüp-fenotüüp seos põhjuslike mutatsioonidega ................................................ 202 Eksperimentaalosa........................................................................................................... 222.1 Töö eesmärgid ............................................................................................................ 222.2 Materjal ja metoodika ................................................................................................. 222.2.1 Valim ................................................................................................................. 222.2.2 GDNF-i sekveneerimine Sangeri meetodil ....................................................... 222.2.3 Andmete analüüs ............................................................................................... 242.2.4 GDNF-i sekveneerimine teise põlvkonna sekvenaatoriga ................................ 252.3 Tulemused ja arutelu .................................................................................................. 262.3.1 Tulemused ......................................................................................................... 262.3.2 Arutelu .............................................................................................................. 28Kokkuvõte ................................................................................................................................ 31Sequencing and analysis of mutations in the GDNF gene in individuals with congenitalkidney anomalies ...................................................................................................................... 32Summary ................................................................................................................................... 32Tänusõnad ................................................................................................................................. 33Kasutatud kirjandus .................................................................................................................. 34Kasutatud veebiaadressid ......................................................................................................... 38Lisad ......................................................................................................................................... 392