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DNA Sequencing  Genetically Inherited Diseases

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DNA Sequencing  Genetically Inherited Diseases DNA, MUTATION DETECTION TYPES OF DNA There are two major types of DNA: Genomic DNA and Mitochondrial DNA Genomic DNA / Nuclear DNA: Comprises the genome of an organism and lead to an expression of genetic traits. Controls expression of the various traits in an organism. Sequenced as part of the Human Genome Project to study the various functions of the different regions of the genome Usually, during DNA replication there is a recombination of genes bringing about a change in sequence leading to individual specific characteristics. This way the difference in sequence could be studied from individual to individual. MITOCHONDRIAL DNA(MT DNA) mtDNA is a double stranded circular molecule. mtDNA is always Maternally inherited. Each Mitochodrion contains about 2-10 mtDNA molecules. mtDNA does not change from parent to offspring (Without recombination) Containing little repetitive DNA, and codes for 37 genes, which include two types of ribosomal RNA, 22 transfer RNAs and 13 protein subunits for some enzymes HUMAN STRUCTURAL GENE 1. Helix–turn–helix 2. Zinc finger 3. Leucine zipper 4. Helix–loop–helix GENE TO PROTEIN Facilitate transport of the mRNA to the cytoplasm and attachment to the ribosome Protect the mRNA from from 5' exonuclease Acts as a buffer to the 3' exonuclease in order to increase the half life of mRNA. TRANSLATION TYPES OF DNA SEQUENCE VARIATION VNTR: Variable Number of Tandem Repeats or minisatellite (Telomeric DNA, Hypervariable minisatellite DNA) ~6-100 bp core unit SSR : Simple Sequence Repeat or STR (short tandem repeat) or microsatellite ~1-5 bp core unit SNP: Single Nucleotide Polymorphism Commonly used to also include rare variants (SNVs) Insertions or deletions INDEL – small (few nucleotides) insertion or deletion Rearrangement (inversion, duplication, complex rearrangement) CNV: Copy Number Variation SNP Allele 1 A U G A A G U U U G G C G C A U U G C A A Allele 2 A U G A A G U U U G G T G C A U U G C A A A Most are “silent” G Intragenic Promoters and other regulatory sequences Introns Exons 5’ and 3’ untranslated regions Coding sequence (~1-2% of genome) COPY NUMBER VARIATION (CNV) Kb to Mb in size (average ~250 Kb) >>2000 known, affect ~12% of human genome ? ~100 / person Role in human disease/normal traits MUTATIONS SILENT SEQUENCE CHANGE (SYNONYMOUS SNP) mRNA Normal A U G A A G U U U G G C G C A U U G C A A Protein Met Lys Phe Gly Ala Leu Gln Sequence mRNA variant A U G A A G U U U G G U G C A U U G C A A Protein Met Lys Phe Gly Ala Leu Gln Changes that do not alter the encoded amino acid Missense Mutation (Nonynonymous SNP) mRNA Normal A U G A A G U U U G G C G C A U U G C A A Protein Met Lys Phe Gly Ala Leu Gln mRNA Missense A U G A A G U U U A G C G C A U U G C A A Protein Met Lys Phe Ser Ala Leu Gln Missense: changes to a codon for another amino acid (can be harmful mutation or neutral variant) Nonsense Mutation (Nonynonymous SNP) mRNA Normal A U G A A G U U U G G C G C A U U G C A A Protein Met Lys Phe Gly Ala Leu Gln mRNA A U G U A G U U U G G C G C A U U G C A A Nonsense Protein Met Nonsense: change from an amino acid codon to a stop codon, producing a shortened protein Frameshift Mutations mRNA Normal A U G A A G U U U G G C G C A U U G C A A Protein Met Lys Phe Gly Ala Leu Gln mRNA Frameshift A U G A A G U U G G C G C A U U G C A A Protein Met Lys Leu Ala Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and (usually) shortened protein Splice-site Mutations Exon 1 Intron Exon 2 Intron Exon 3 Exon 2 Exon 1 Exon 3 Altered mRNA Splice-site mutation: a change that results in altered RNA sequence CHROMOSOMAL DISORDERS 50% of 1st trimester miscarriages 5% of stillbirths 0.5% of liveborns Down syndrome—trisomy 21 Fragile X syndrome Somatic cell abnormalities in cancers Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. There are many types of chromosome anomalies. They can be organized into two basic groups, numerical and structural anomalies. NUMERICAL ABNORMALITIES Loss or gain of one or more chromosomes, referred to as aneuploidy The addition of one or more complete haploid complements, known as polyploidy STRUCTURAL ABNORMALITIES Deletions: A portion of the chromosome is missing or deleted. Duplications: A portion of the chromosome is duplicated, resulting in extra genetic material Translocations: A portion of one chromosome is transferred to another chromosome. There are two main types of translocations: Reciprocal translocation: Segments from two different chromosomes have been exchanged. Robertsonian translocation: An entire chromosome has attached to another at the centromere - in humans these only occur with chromosomes 13, 14, 15, 21 and 22 STRUCTURAL ABNORMALITIES Inversions: A portion of the chromosome has broken off, turned upside down and reattached, therefore the genetic material is inverted and sequence is disturbed Paracentric: involves only one arm of the chromosome Pericentric: segment involves the centromere Insertions: A portion of one chromosome has been deleted from its normal place and inserted into another chromosome. Rings: A portion of a chromosome has broken off and formed a circle or ring. This can happen with or without loss of genetic material. Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. STRUCTURAL ABNORMALITIES RECIPROCAL TRANSLOCATION ROBERTSONIAN TRANSLOCATION CHROMOSOME NOMENCLATURE MOSAICISM & CHIMERISM Mosaicism:The presence in an individual, or in a tissue, of two or more cell lines that differ in their genetic constitution but are derived from a single zygote Chimerism:The presence in an individual of two or more genetically distinct cell lines derived from more than one zygote Dispermic Chimeras: two genetically different sperm fertilize two ova and the resulting two zygotes fuse to form one embryo. Blood Chimeras: result from an exchange of cells, via the placenta, between non-identical twins in utero METHODS OF CHROMOSOME ANALYSIS Patient cells are incubated and divide in tissue culture. Phytohemaglutinin (PHA): stimulates cell division Colcemid: arrests cells in metaphase 3:1 Methanol:Acetic Acid: fixes metaphase chromosomes for staining Giemsa-, reverse- or centromere-stained metaphase chromosomes PREPARATION OF A KARYOTYPE G-BANDED METAPHASE SPREAD KARYOTYPE FLUORESCENT IN SITU HYBRIDIZATION (FISH) . Hybridization of complementary gene- or region- specific fluorescent probes to chromosomes. Interphase or metaphase cells on slide (in situ) Probe Microscopic signal (interphase) USES OF FLUORESCENT IN SITU HYBRIDIZATION (FISH) Identification and characterization of numerical and structural chromosome abnormalities. Detection of microscopically invisible deletions or duplication. Detection of sub-telomeric aberrations. Prenatal diagnosis of the common aneuploidies (interphase FISH). FISH PROBES Chromosome-specific centromere probes (CEP) Hybridize to centromere region Detect aneuploidy in interphase and metaphase Chromosome painting probes (WCP) Hybridize to whole chromosomes or regions Characterize chromosomal structural changes in metaphase cells Unique DNA sequence probes (LSI) Hybridize to unique DNA sequences Detect gene rearrangements, deletions, and amplifications CENTROMERIC PROBES (Ch 13 red, Ch18 pink, Ch 21 green, X yellow, Y white) LOCUS-SPECIFIC PROBES Ch 15 centromere (green) Ch 15 PWS critical region (red) CHROMOSOME PAINTING PROBES (CH 9 GREEN, DER CH 10) CHROMOSOME PAINTING PROBES MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA) • Gold standard for DNA copy number quantification • Also be applied to investigate the methylation status of DNA sequences • Popular applications include: • Predisposition to Cancer • Neuromuscular Disorders • Intellectual Disability • Solid Tumours DETECTION OF X CHROMOSOME COPY COMPARATIVE GENOMIC HYBRIDIZATION (ARRAY CGH) CHROMOSOMAL ABNORMALITIES WITH ARRAY CGH COMPARISON BETWEEN DIFFERENT METHODS FOR THE DETECTION OF GENE DELETIONS/DUPLICATIONS POLYMERASE CHAIN REACTION (PCR) USES FOR PCR Clinical Research DNA fingerprinting Gene cloning Crime scene analysis Paternity testing Real-time PCR Archeological finds DNA sequencing Genetically inherited diseases RFLP (RESTRICTION FRAGMENT LENGTH POLYMORPHISMS) • RFLP is an enzymatic procedure for separation and identification of desired fragments of DNA. • Using restriction endonuclease enzymes fragments of DNA is obtained and the desired fragment is detected by using restriction probes. • May be used to differentiated two organism by analysis of patterns derived from cleavage of their DNA. • Variations commonly result in RFLPs: • Single-base changes in the nucleotide sequences (SNP), • Tandem repeats (VNTR), • Polymorphisms, • Mutations. • SNP or VNTR, are simply markers, which, in most cases, have no known effect on the structure or rate of production of any particular protein. APPLICATION OF RFLP TEST Paternity test Criminal investigation To detect mutated gene QUANTITATIVE REAL TIME PCR QRT-PCR ANALYSIS WESTERN BLOTTING WB RESULTS DNA SEQUENCING AND ITS TYPES PCR Insect DNA extraction identification ? Bioinformatics Gel electrophoresis DNA sequencing ACAGATGTCTTGTAATCCGGCCGTTG GTGGCATAGGGAAAGGACATTTAGT GAAAGAAATTGATGCGATGGGTGGA TCGATGGCTTATGCTATCGATCAATC AGGAATTCAATTTAGAGTACTTAAT AGTAGCAAAGGAGCTGCTGTTAGAG CAACACGTGCTCAGGCAGATAAAAT ATTATATCGTCAAGCAATACGTAGT ATTCTTGAATATCAAAAATTTTTGT TGGTTATTCA DNA SEQUENCING Probably the
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