TIDAK SEMUA MUTASI PADA LOKUS RESTRIKSI PCR/OLA POLYMERASE CHAIN REACTION/ OLIGONUCLEOTIDE LIGATION ASSAY MUTASI PADA 106 A:T KE G:C TARGET DIAMPLIFIKASI –PCR HIBRIDISASI : PELACAK X DAN Y LIGASI
PCR/OLA Like sickle cell anemia many genetic diseases are caused by mutant genes. E.g.? Many diseases are caused by a single nucleotide (nt) change in the wild type gene. A single nt change can be detected by PCR/OLA ( oligonucleotide ligation assay). E.g. The normal gene has A at nt position 106 and mutant has a G. 2 short oligonucleotides (oligo) are synthesized Oligo 1 (probe x) is complementary to the wild type has A at 106 (3’ end).
PCR/OLA Oligo 2 ( probe y) has G at 107 (5’ end). The two probes are incubated with the PCR amplified target DNA. For the wild type the two probes anneal so that the 3’end of probe x is next to the 5’end of probe y. For the mutant gene the nt at the 3’ end of probe x is a mismatch and does not anneal.
PCR/OLA DNA ligase is added. The two probes will only ligate if the two probes are perfectly aligned (as in the wild type). To determine if the mutant or wild type gene is present it is necessary to detect for ligation. Probe x is labeled at 5’ end with biotin Probe x is labeled at 5’ end with digoxygenin.
PCR/OLA Digoxygenin serves as an antibody binding indicator. After washing a colourless substrate is added. If a coloured substrate appears this is indicative that the biotin probe (x) ligated to the dioxygenin probe (Y) and that the wild type gene is present.
Enzymes as Therapeutic Agents/ DNase1 Cystic fibrosis (CF) is one of the most fatal heredity diseases among European and their descendants with ~30,000 cases in the US and 23,000 in Canada. Furthermore among European descendants it occurs in 1 in 2,500 live birth and 1 in 25 are carriers. It is caused by more than 500 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Individuals with CF are highly susceptible to bacterial infection and antibiotic treatment often results in resistant strains.
DNase 1 A thick mucus which is a results of: Alignate produced by bacteria DNA from lysed cells Leucocytes which accumulate due to the infection Makes breathing difficult. Scientist at Genentech isolated the gene for DNase1 The purified enzyme was delivered as an aerosol to the lung where it hydrolysed the DNA into short oligionucleotides. This decrease the viscosity in the lungs and made breathing easier.
Alginate Lyase Alginate is a polysaccharide polymer that is produced by seaweed and some soil and marine bacteria. The excretion of alginate by Pseudomonas aeruginosa of patients with CF contributes to the viscosity in the lung. The enzyme alginate lyase can liquefy bacteria alginate. Alginate lyase was isolate from Flavobacterium sp. and cloned into E. coli.
Aliginate Lyse The expressed gene produced a protein of 69,000 Da. The 69,000 Da protein produced a proteolytic enzyme of 6,000 Da. The remain 63,000 Da protein was cleaved to produce a 43,000 Da which is able to liquefy bacterial alginate. Combined with DNase1, alginate lyse is able to reduce the mucus in the lungs of patients with CF.
THE SOLUTION ? Current laboratory techniques not entirely satisfactory Need trained staff, equipment, slow throughput Rapid molecular tests being developed
ENZYME BASED simple technique. large number of typing enzymes available many samples typed at same time power to distinguish morphologically similar parasites. Significant tissue needed for analysis visceral leishmaniasis requires spleen, liver, Technique not rapid can take days Sometimes incorrect diagnosis enzyme labile Technique simple but equipment expensive (+) (-)
Iso-enzymes separated by charge: Isoelectric focusing equipment
rapid easy field based tests can be developed useful for both individual & mass population screening cannot distinguish past/ present infections cannot distinguish morphologically similar parasites expensive to develop significant research prior to commercialization (+) (-) ANTIBODY BASED
DNA BASED Nonculturable agents Human papilloma virus Hepatitis B virus Fastidious, slow-growing agents Mycobacterium tuberculosis Legionella pneumophilia Highly infectious agents that are dangerous to culture Francisella tularensis Brucella species Coccidioidis immitis
DNA BASED In situ detection of infectious agents Helicobacter pylori Toxoplasma gondii Agents present in low numbers HIV in antibody negative patients CMV in transplanted organs Organisms present in small volume specimens Intra-ocular fluid Forensic samples
RRestriction endonuclease analysis PPCR DDNA Hybridization DDNA fingerprinting DNA BASED