molecular pathology testing of nucleic acids within a clinical context helpful hereditary disorders...

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  • Molecular Pathology Testing of nucleic acids within a clinical context Helpful Hereditary disorders Oncology Infectious diseases
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  • Molecular Pathology Specific purposes Diagnosis Prognosis Prenatal testing Pharmacotherapy Pharmacogenetics Pharmacogenomics
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  • Watson and Crick The structure of DNA was described by British Scientists Watson and Crick as long double helix shaped with its sugar phosphate backbone on the outside and its bases on inside; the two strand of helix run in opposite direction and are anti-parallel to each other. The DNA double helix is stabilized by hydrogen bonds between the bases Doctortvraos e learning series
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  • Watson and Crick discovers DNA / Feb 28 th 1953
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  • Watson and Crick Builds a Model DNA 7 th March 1953
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  • First Document on DNA published in Nature 25 th April 1953
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  • Watson and Crick - Awarded Nobel Prize in 1962
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  • DNA A molecule contains two polynucleotide strands that form an an antiparallel double helix. Nucleotides: Nitrogenous base (AT GC,U) Deoxyribose Phosphate
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  • DNA - Structure The nucleotide, however, remains as the fundamental unit (monomer) of the nucleic acid polymer. There are four nucleotides: those with cytosine (C), those with guanine (G), those with adenine (A), and those with thymine (T).
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  • DNA A purine always links with a pyrimidine base to maintain the structure of DNA. Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them. Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.
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  • Chemical structure of DNA
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  • DNA is Endless structure The rungs of the ladder can occur in any order (as long as the base- pair rule is followed) Those 4 bases have endless combinations just like the letters of the alphabet can combine to make different words.
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  • DNA Example First strand GGGTTTAAACCC Second strand CCCAAATTTGGG
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  • Central Dogma of Molecular Biology
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  • DNA makes a Copy of Self Replication is the process where DNA makes a copy of itself. Why does DNA need to copy? Simple: Cells divide for an organism to grow or reproduce, every new cell needs a copy of the DNA or instructions to know how to be a cell. DNA replicates right before a cell divides.
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  • DNA Replication DNA replication is semi-conservative. That means that when it makes a copy, one half of the old strand is always kept in the new strand. This helps reduce the number of copy errors. So we remained what we were ?
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  • Transcription RNA polymerase II mediates transcription and generates a precursor ss-mRNA identical to the sense (coding) stand except for U for T. Precursor ss-mRNA is processed in nucleus by spliceosomes that catalyze intron removal and exon ligation with the regulation by exonic and intronic enhancers and silencers with production of different pre m-RNA that go across nucleus
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  • DNA to RNA creates functional translations DNA remains in the nucleus, but in order for it to get its instructions translated into proteins, it must send its message to the ribosome's, where proteins are made. The chemical used to carry this message is Messenger RNA Doctortvraos e learning series
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  • DNA RNA DNA a never ending cycle RNA has the job of taking the message from the DNA to the nucleus to the ribosome's. Transcription - RNA is made from DNA Translation - Proteins are made from the message on the RNA Doctortvraos e learning series
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  • Translation m-RNA directs protein synthesis. Occurs in ribosomes (rRNA+proteins) Codons (three bases) are read by transfer (tRNA) There are 64 possible codons, therefore most of the 21 aminoacids are specified by more than 1 codon
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  • RNA = Ribonucleic acid. RNA is similar to DNA except: It has one strand instead of two strands. Has uracil instead of thymine 3.Has Ribose instead of Deoxyribose
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  • Gene Expression DNA level expression control Transcriptional Post-Transcriptional Epigenetics DNA methylation Histone modification
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  • Gene Expression DNA level expression control Transcriptional House keeping genes Always on Transcription factors Usually lie upstream in the promoter region Enhancer and silencer elements
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  • Gene Expression Post transcriptional Export of mRNA out of nucleus Alternative splicing mRNA stabilization mRNA degradation RNA interference or silencing miRNA and siRNA
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  • Gene Expression DNA level expression control Transcriptional Post-Transcriptional Epigenetics DNA methylation Histone modification
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  • What is Gene The gene, the basic units of inheritance; it is a segment within a very long strand of DNA with specific instruction for the production of one specific protein. Genes located on chromosome on it's place or locus.
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  • Modes of inheritance Mutations of single genes Patterns: autosomal dominant, autosomal recessive, X- linked, mitochondrial Anticipation Increased severity of a certain diseases in successive familiar generations associated with triple repeats Mosaicism At least two cell lines derived from a single zygote
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  • Modes of inheritance Genomic imprinting Different expression of alleles depending on parent origin Uniparental Disomy Both copies inherited from one parent Environmental influence Chronic diseases
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  • Mutations and Polymorphisms Mutation: change in DNA sequence Polymorphism: non disease causing change in DNA or a change found at a frequency of 1% in population When evaluating changes in DNA sequence use neutral terms: sequence variant, sequence alteration or allelic variant. There may be: Missense, nonsense, deletions, insertions, frame shifts, duplications, amplifications, trinucleatide repeats.
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  • Single Nucleotide Polymorhisms and Haplotypes SNPs are single base differences in the DNA of individuals There are ~10 million SNPs in the human genome IMPORTANCE: Pharmacogenetics Ex. CYP (cP450) Alleles of SNPs that are close together tend to be inherited together. Haplotype: a set of associated SNPs alleles in a region of a chromosome
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  • Overview of Molecular Techniques and Instrumentation Standard or usual specimen flow Specimen collection (blood, tissue) Nucelic acid isolation (DNA or RNA) Nucleic acid quantification (optional) Nucleic acid storage Nucleic acid amplification (or other) Test interpretation Quality control
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  • Nucleic acid isolation (DNA or RNA) Manual vs. automated Cell lysis Dependent of specimen type, nucleic acid being isolated for, desired purity and application to be used in FFPE yields ~200 pairs Purification Organic: phenol-chloroform Non organic: silica, anion exchange chromatography and magnetic particles DNA or RNA Isolation RNA rapidly degrades
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  • Methods DNA sequencing Southern Blot PCR RT-PCR Real Time PCR Methylation-Specific PCR In-situ PCR Protein Truncation Test Transcription-Mediated Amplification Strand Displacement Amplification Nucleic Acid Sequence- Based Amplification Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
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  • Gene sequencing Determining the exact sequence of the four bases in a given DNA template Two methods Maxam-Gilbert Chemical degradation Sanger Chain termination Radiolabeled, Dye-prime or Dye-terminator (cycle sequencing) Pyrosequencing Sequnces a short length of DNA (~30-60 bases)
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  • Applications of Direct DNA sequences Clinical conditionGene HIV drug resistanceHIV-protease, RT Cystic fibrosisCFTR gene Beta thalassemiaBeta globin Cancer predisposition breastBRCA1 Hereditary non polyposis colon cancer TP53 MENPTEN Ret proto-oncogene Congenital hearing lossConnexin 26 HCV genotyping5UTR
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  • Array-based Comparative Genomic Hybridization Comparative Genomic Hybridization is done in metaphases in classical cytogenetics (M-CGH) Resolution 5 Mb Bacterial Artificial Chromosome (BAC) maps the human genome therefore an Array based-CGH can be created (A- CGH). Different resolutions up to 32,000 (45 kb) cDNA-CGH Oligonucleotide-CGH Can detect Single Nucleotide Pleomorphisms (SNPs) [Gene Chip]
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  • Methods DNA sequencing Southern Blot PCR RT-PCR Real Time PCR Methylation-Specific PCR In-situ PCR Protein Truncation Test Transcription-Mediated Amplification Strand Displacement Amplification Nucleic Acid Sequence- Based Amplification Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
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  • Southern Blot Edwin M South

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