Adobe Systems Metody detekce nukleotidových polymorfismů Andrea Knight, PhD ÚPF Lékařská fakulta MU Point mutation vs polymorphism •Both are single nucleotide differences in DNA sequence •Point mutation: •Polymorphism: –a DNA sequence at a certain locus that varies among individuals (is present as two or more alleles) –any sequence variant present at a frequency of > 1% in a population Types of polymorphisms •single nucleotide polymorphism (SNP) •short tandem repeats (STR), microsatellites –Repeat length 2 -5 bps •variable number of tandem repeats (VNTR) or minisatellites –Repeat length 10 - 200 bps •restriction fragment length polymorphisms (RFLPs) •insertion/deletion To be classified as a SNP: Two or more versions of a sequence must each be present in at least 1% of the general population. SNP Single nucleotide polymorphisms •Nucleotide: A, T, C, G •SNP may replace the nucleotide cytosine (C) with the nucleotide thymine (T) •occurs once in every 300 nucleotides •there are ~ 10 million SNPs in the human genome –Human genome completed & published: 1st draft April 2003, May 2006 (incl. chr.1) •most commonly found in DNA between genes •when SNPs occur within a gene or in a regulatory region near a gene, they plays a direct role in disease by affecting the gene’s function Differences in SNPs and disease-causing mutations Population Distributions Linked SNPs: -do not reside within genes -do not affect protein function -do correspond to a particular drug response or to the risk for getting a certain disease Causative SNPs: -affect the way a protein functions -correlating with a disease or influencing a person's response to medication Polymorphisms •are harmless/non-pathogenic sequence variants •are useful for: •forensic analysis (DNA profiling) •mapping disease genes •inheritance of disease genes in the family •as predictive markers of effectiveness to drugs •are now intensively studied https://www.ncbi.nlm.nih.gov/snp/ • •Databases of SNPs contains human single nucleotide variations, microsatellites, insertions and deletions, population frequency, molecular consequence, genomic and RefSeq mapping information for both common variations and clinical mutations. SNP example - Sickle cell anaemia –Impaired production of red blood cells (RBC) –Inheritance of two abnormal Βeta-globin gene (chr 11) –Estimated that 7% of world's population (~420 million) are carriers –The gene defect is a SNP, where GAG codon changes to GTG and results in glutamic acid being substituted by valine (E6V) 17-10_NrmlSicklCompr_1 Polymorphism of regulatory gene •IRF5; interferon regulatory factor 5 •=transcription factor •Associated with increased risk of SLE •In type-I diabetes •In melanona •Correlates with acute rejection of liver transplant Metody detekce SNPs •PCR •Real-time PCR •RFLP –ELFO na agaróze •PAGE –SSCP, heteroduplexní analýza •Kapilární elektroforéza –v podstatě varianta PAGE Strategie volby metody •Flexibilita •Cena •Dostupnost •Rychlost Polymerase Chain Reaction •1983 Kary Mullis •1993 Nobel Prize • • kary PCR is a DNA synthesis •DNA primers – complementary to the sequences that flank the region to be amplified •Deoxynucleotides (dNTPs) –dATP+dTTP+dCTP+dGTP •Mg2+ co-factor for enzyme •Buffer •DNA polymerase – heat resistance Taq polymerase –Thermophilus Aquaticus – synthesis PCR •PCR is DNA synthesis •First strands are separated (denaturation) •Heat to 95ºC •DNA primer instead of RNA primer to provide free 3’ OH •DNA primers designed to be complementary to sequences that flank the region to be amplified •Primers hybridize to DNA (anneal) •DNA is synthesised (extension) •Use heat resistant DNA polymerase – Taq polymerase • • Reakce •96ºC Iniciální denaturace •96ºC denaturace •40-72ºC annealing •72ºC elongace •72ºC závěrečná elongace •4ºC zchlazení 30-40x Agarose gel electrophoresis Animation2 + - Agarose gel electrophoresis: discuss onk_ban_462_492_2 PCR s následnou RFLP •Pokud polymorfismus způsobuje vznik restrikčního místa: –podrobíme produkt PCR působení daného enzymu a na agarové elektroforéze pozorujeme výsledek – •Mismatch primery – umožňují vnést restrikční místo do blízkosti SNP a umožní jeho detekci pomocí endonukleáz PCR s analýzou délky produktů •Pokud je polymorfismus způsoben insercí/delecí většího počtu bází: –produkt PCR analyzujeme přímo na agarové elektroforéze • •V případě analýzy dinukleotidových repetic používáme kapilární elektroforézu • •Alelově specifické primery – poskytnou produkt jen za přítomnosti dané alely PCR Question #1 0 10 15 20 25 30 40 cycles Agarose gel electrophoresis Explain the intensity of the bands as the number of PCR cycles increases PCR Question #2 45° 55° 60° 65° Agarose gel electrophoresis Explain the pattern of the bands as annealing temperature is changed Real-time PCR •Různé systémy sond (TaqMan, FRET) •Melting curve analysis Kvantitativní vztah mezi : množstvím PCR produktu (amplikonu) a intenzitou fluorescence • • Amplifikační práh detekce (Ct) Real – time detekce amplifikace Plateau fáze Exponenciální amplifikace pod úrovní pozadí Exponenciální fáze „end point“ „real-time“ Threshold cycle „Ct“ – určený na základě hodnoty fluorescence pozadí (backround) a aktuální fluorescence vzorku – kvantitativní výstup pro každý vzorek Real – time detekce amplifikace - Ct Threshold Threshold cycles Background Fluorescence Jak předejít kontaminaci Kontaminace • Správná laboratorní praxe • Plastik v RNA kvalitě • Automatizace ´ www.millipore.com; www.appliedbiosystems.com Application note: Contamination-pipetting: relative efficiency of filter tips compared to Microman® positive displacement pipette. Nature Methods 3 June 2006 Shrnutí •Co jsou SNPs •Metody detekce •Praktikum •