Lab Tests an Diagnosis

LAB TESTS AN DIAGNOSIS Molecular diagnostics • Use of molecules (DNA, RNA and proteins) to assess/diagnose the state of health/disease in a person • Tell if a person is: o Predisposed to a specific disease o Has a specific disease o Has a specific infection (virus or bacteria) o Expected to respond to a specific treatment • E.g. prenatal screening to see if a foetus has abnormal chromosomes (trisomy 21) DNA • • • • Hereditary material in cells determining how our body will develop and function Information is stored in 4 bases A, T, G, C (base pairs: A with T, G with C) which form nucleotides (base + sugar + phosphate) forming two strands of the double helix o E.g. blue eyes = ATCGTT and brown eyes = ATCGCT 3 billion bases and over 20,000 genes on 23 pairs of chromosomes Genes = units of DNA that code for RNA → translated to proteins Reference Sequence • Human Genome Project (HGP) was the international collaborative program to map all genes of humans • First draft came in 2001 and complete sequence published in 2004 • About 20,500 genes were revealed • Took 13 years (1990-2003) and costed USD 2.7 billion • Currently a human genome can be sequenced in few days for ~$1,000 Polymerase Chain Reaction (PCR) • Amplifies specific DNA sequences and produces multiple copies to allow detection/measurement • First step in determining variations in DNA sequence • Carried out in many cycles in a PCR Thermocycler • Requires: DNA template, Primer, dNTPs, DNA Polymerase (Taq), buffer Gel Electrophoresis (separate PCR products) • Gel electrophoresis separates DNA molecules by size/length • When an electric current is applied, negatively charged DNA moves • towards positively charged electrode • Shorter molecules move faster and farther than longer molecules • Size of DNA fragments in sample is compared to a DNA marker (fragments of known length) Whooping cough • 3 year old child has a bad cough and the mother informs that there has been few cases of whooping cough at the childcare centre. You suspect this child also has whooping cough. • Whooping cough is a highly contagious respiratory infection • characterised by severe coughing spells with a whooping sound • Caused by bacteria Bordetella pertussis • PCR to test for whooping cough: o Sample collection – nasopharyngeal (NP) swab o Extract DNA o PCR with primers specific for B pertussis o Gel electrophoresis Quantitative PCR – SYBR green assay • SYBR green binds double stranded DNA (not single stranded) • Emits fluorescence upon binding • Fluorescence intensity correlates with DNA amplified • ↑ PCR → ↑ DNA products → ↑ fluorescence intensity • Quantitative PCR results: • PCR in clinics: o Diagnostic test for: tuberculosis, chlamydia, gonorrhea o Viral type determination for: Human papillomavirus (HPV) o Screening donated blood for: HIV or hepatitis o Quantitative PCR: effectiveness of antiviral therapy by monitoring HIV viral load (measure the amount of HIV RNA copies in the blood) Genetic tests • Detect DNA variations that may cause disease • Different methods depending on size of variations o Fluorescent in situ hybridization (FISH) o DNA microarray (array CGH) o DNA sequencing • Types of variations o Large: CNV o Small: SNP Detecting large variations • Cytogenetics – the study of chromosomes • Karyotyping – chromosome stained and viewed under microscope • Fluorescent in Situ Hybridisation (FISH) – fluorescent probes bind to DNA sample + viewed under microscope DNA microarrays (array CGH) • Detects extra or missing segments of DNA • Copy number variation • Detects gains or losses that are smaller (<200kb) • Combines Comparative Genomic Hybridization (CGH) and DNA microarray (array CGH) • Normal: two copies of every gene in each cell Abnormal: > two or < two copies of genes • Clinical use: o Unexplained problems in children: learning, physical development, behavior, birth defects e.g. mental retardation, developmental delay, autism, congenital abnormalities o Prenatal screening e.g. trisomy 21 o Genetic abnormalities in cancer