MUNI MED Genetics in Dentistry - Practice Ing. Hana Holcova Polanská, Ph.D. Mgr. Lucie Válkové Methods in Molecular Biology I Biological Materials Biological material is everything that was or is a part or a product of a living organism - dried herbal tea mixture - apple core - oak plank - cat droppings/fur - tube with SARS-CoV-2 virus - bodily fluids - urine, blood, plasma, serum, spit, ejaculate, phlegm - tissues, cells Methods in Molecular Biology - Determination of nucleic acids • PCR, RFLP-PCR - detection by ELPHO • Real time PCR • Sequencing - Determination of proteins • EUSA • Western blot • other methods based on antigen-antibody interactions - Other molecular biology methods Nucleic Acid Isolation In native state from native material - in sufficient quantity and required purity. NAs need to be devoid of all substances that would after lysis become a part of the crude substrate and that would impair the specific effects of enzymes used for further analyses. Homegenizatton ^3 Lysis Isolation of genomic DNA Isolation of RNA - focus on protection against degradation! Homogenization/ Lysis Separation V Phase Separation Extraction/ Precipitation Precipitation Resuspension I Guanidinium-phenol Water/TE UNA p. CI Methods in Molecular Biology II PCR - Polymerase Chain Reaction Aim - acquisition of required specific sequence of genomic DNA without previous cloning Principle - multiple replications • 25 to 35 cycles • depends on temperature of reaction mixture • amount of replicated DNA grows exponentially (2n) PCR Protocol Get the reagents Prepare the mix Set up conditions Thermocycler ^2 Analyze the gel Negative result 13 SfeMii«^ *it«\ce PCR Multiple in vitro replication in a tube Chain reaction based on DNA replication Repeating cycles: - denaturation (separation of dsDNA) - 96 °C - annealing - primer binding - 50-65 °C - elongation - synthesis of a new DNA strand - 72 °C Denature DNA sample to separate DNA strands {94°C, 5 min) Primers bind to DNA strands (30-65°C, 30 s] Denature to separate DNA strands (94°C, 30 s) Polymerase synthesizes new DNA strands (72'C, 45 s-2 min] Region o(^~~ " interest 3' I I I I I I I I I I I I I I I I I I I ' ' ' '........I I I 1 I I I I 98°C II II I I I I I I I II I I I I I I I I I I I I I I I I I I I 5' 48 to 72°C Denaturation 'temperature is increased to separate DNA strands Primer 1 1 1 1 1 1 1 1 1 1......-v Annealins 3" -L 5' Temperature is decreased Primer 1 'I 1 1 1 1 1 1 I 1 1 1 1 1 1:. I 68 to 72°C to allow primers to base pair to complementary DNA template TTTT" ¥^-rTTT-' I I I I I I I I I II I I I I I I 5'"^ Extension Polymerase extends primer to form nascent DNA strand 1st cycle — 111HIH! I! H11ITTT lllllllllllllllllll 22 = 4 copies 2nd cycle rmrni I1INI1I !!!!!!!! 1....... ! ........ anno 1 lull!! tiiiiiii I V = 8 copies 3rd cycle — initiiimimrm-..........I .....1...........n i!ii:iiiiiiin;:!ii ""»"".......rrr ............m '.......1.......rn iiiiiimiiiniim 24 = 16 copies 4th cycle .....11111111111 IIIIIIIIIIIIIIITT limimHIHHI iniiiiiiiiiiiii: "I"""......11 .....'........." .....1.........'I H............... IIIIIIIMIIIIIIII lilllllilliill llllllllllllllllll iniiiiiiiiiiiiin ->■ 30th cycle 231 = 2 billion copies Exponential Amplification Process is repeated, and the region of interest is amplified exponentially IIIIIIIIIIM111 i 11 25 = 32 copies PCR DNA replication - in vitro (PCR) • DNA polymerase • thermostable (resists to temperatures up to 98 °C) • Taq [Thermus oquoticus), Tth [Thermus thermophilus) • primer • short specific segments of DNA • oligonucleotide 20-25 pb • limiting the region for DNA amplification • Mg2+ ions • affect activity and precision of polymerase • template DNA dNTP buffer (pH=8) temperature DNA TTTTTITT iririmn * Primers 0s »„ } DNA Polymerase DNA replication - in vivo • enzymes - helicase, primase, DNA polymerase, ligase... DNA replication DNA polymerase C) Requires radioactive labelling on one end of ssDNA. Reaction is done in 4 tubes - in each only some types of bases are cleaved. Mixture of differently long fragments ending in a place of a specific base is created —► evaluation using ELPHO, determination of a sequence of a given section. I I I I I I I I I OA A>G T+C G>A A>G T+C 1 I I 1 I I 1 I I ACAATGCGT Single stranded DNA template Cleave at specific bases Separate by gel electrophoresis antl detect labels T G c G T A A C A Video: https://www.youtube.com/watch?v= B5DJ8PL4E0 Sanger Sequencing Enzymatic method Based on principle of replication - end of DNA synthesis in the moment ddNTP is incorporated instead of dNTP ddNTP = analogue of dNTP, but lacks 3'-OH group on carbon ddNTP - terminator Reaction mixture (4x) • DNA template • primer • Taq DNA polymerase - synthesis of DNA from 5' to 3' end • buffer • dNTP - abundant (to get fragments of all possible lengths) • ddNTP - low concentration Evaluation - electrophoresis Modification -> fluorescently labeled ddNTP (4 different color labels) - reaction performed in one tube x i i I 1 i i i i I / I|2|3|4|5I6I7I8:J 10 DNA to be sequenced TGGTCGACGA I I I I !|2|3|4|5|6|7 i t 10 Replication-stopping analogue in the X" reaction mixture Complementary strands produced in the "C" reaction A C C A G C ACCAGCTGC í |2 |3 U I 5 |« I? í i I i Video pro názornost: https://www.voutube.com/watch?v=wdS3iOTgbiM Sanger Sequencing Capillary sequencing of DNA with fluorescently labeled ddNTP ® Reaction mixture ► Primer and DNA template ► DNA polymerase ► ddNTPs with flourochromes ► dNTPs (dATP, dCTP, dGTP, and dTTP) Primer rjl' | | | | | | | | | 3' 5't-t S'-r-r 5't-t 5't- 5T S'l I I I "..........1" Template ddNTPs ddTTP -• ddCTP-# ddATP-ddGTP- ® Primer elongation and chain termination .......T 3' ........., 3' «4-o4-V*«W»» Oil 0 0 0 ho— J>-c—^-o—o. Ah oh oh @ Capillary gel electrophoresis separation of DNA fragments 1 Laser detection of flourochromes and computational sequence analysis Cliromalograph NGS - Next Generation Sequencing Sequencing of thousands to millions of sequences at the same time Template DNA are fragmented sections few hundred bases long Ends of fragments are enzymatically blunted and connected to oligonucleotides of specific sequence (= adapters) Single fragments are separately amplified via PCR and in the next step sequenced in parallel Fragmented input DNA End Repair dA Tailing Adaptor Ligation Size Selection Amplification Next Generation Sequencing Use: - whole genome sequencing - sequencing of chromosomes, plasmids, mt - study of genetic variability mutational analysis - transcriptome analysis Video: https://www.youtube.com/watch? v=shoje 9IYWc https://www.youtube.com/watch? v=CZeN-lgjYCo https://www.youtube.com/watch? v=fCd6B5HRaZ8 Western Blot • Qualitative or semi-quantitative detection of proteins • Principle - detection of protein in gel in electric field utilizing antigen-antibody bonding • 3 phases 1. Electrophoretic separation of proteins (Polyacrylamide gel) 2. Transfer of separated proteins 3. Detection of proteins Practical Part Practical Part 1. PCR 2. ELPHO 3. Evaluation of ELPHO Practical Part 1 Practical implementation of PCR volume in microtube: 25 |iL each sample Composition (1 sample): 2 Primers 10 pmols (1.25 ui) MgCI2 25mM (4 ui) dNTP mix (0.5 ui) Taq polymerase 1U (1 ui) Buffer DYNEX (2.5 ui) PCR H20 (12.5 ui) Template DNA 50ng (2ui) 1 drop of mineral oil 1. 95°C.....5 minutes 2. 95°C.....1 minute 1 3. 60°C.....1 minute 4. 72°C.....1 minute 5. 72°C.....7 minutes 6. 10°C.....10 minutes Practical implementation of PCR 1) Prepare Master Mix for all samples you have by multiplying prescription by the number of samples (plus 1 extra sample). 2) Divide Master Mix to each microtubes by 23 [il. 3) Add one sample (2 piL) to each microtube. Primer A Primer B MZSUL 1.25UL Li. Taq dNTP mix polymerase Buffer 0.5 UL 1 uL PCR water 12.5 uL I <4- MasterMix Divide Master Mix to each microtubes by 23 uL. Multiply prescription by the number of samples plus 1 extra sample {for 5 samples multiply by 6) add 2 uiL sample: -j Created in BioRender.com bio How to Use a Micro pipette Practical Part 2 Practical implementation of ELPHO 1. Prepare casting tray - combs and tape 2. Prepare gel (2%): weigh agarose and add it to Erlenmeyer flask, add TBE buffer (200 ml) 3. Weigh and boil in microwave. 4. After cooling to cca 40 °C add EtBr (1 nl/10 ml) 5. Cool down the gel (cca 30 min) 6. Remove combs and put into ELPHO container with TBE buffer (electrolyte) 7. Add size standard ladder (DNA + dye) 8. Prepare drops of loading dye on paraffin paper and mix with DNA samples. 9. Load samples into wells (max 15 u.1) 10. Connect to power supply 11. Track the progress of DNA through the gel (40 min) 12. Visualisation under UV and evaluation GeneRuler™ 100 bp DNA Ladder O'GeneRuler'" 100 bp DNA Ladder, ready-to-use bp IIU'0.5 |M % bp 1/1000 45.0 9.0 16 900 45.0 9.0 ■ 800 ■ 700 m 45.0 9.0 45.0 9.0 45.0 9.0 500 115.0 23.0 400 40.0 8.0 40.0 8.0 I- 300 I- 200 . 1000 900 ■ 800 • 700 • 600 500 40.0 8.0 ■ 100 40.0 8.0 0.5 Ma-'lane. 8 cm length gti. 1XT6E,5V/cm.1 h 5 0.5 pgflane, 20 cm length pel, 1XTAE,8Wcm,3h Practical implementation of ELPHO 1. Prepare casting tray - combs and tape 2. Prepare gel (2%): weigh agarose and add it to Erlenmeyer flask, add TBE buffer (200 ml) 3. Weigh and boil in microwave. 4. After cooling to cca 40 °C add EtBr (1 u.l/10 ml) 5. Cool down the gel (cca 30 min) 6. Remove combs and put into ELPHO container with TBE buffer (electrolyte) 7. Add size standard ladder (DNA + dye) 8. Prepare drops of loading dye on paraffin paper and mix with DNA samples. 9. Load samples into wells (max 15 u.1) 10. Connect to power supply 11. Track the progress of DNA through the gel (40 min) 12. Visualisation under UV and evaluation GeneRuler™ 100 bp DNA Ladder O'GeneRuler'" 100 bp DNA Ladder, ready-to-use bp IIU'0.5 |M % bp 1/1000 45.0 9.0 16 900 45.0 9.0 ■ 800 ■ 700 m 45.0 9.0 45.0 9.0 45.0 9.0 500 115.0 23.0 400 40.0 8.0 40.0 8.0 I- 300 I- 200 . 1000 900 ■ 800 • 700 • 600 500 40.0 8.0 ■ 100 40.0 8.0 0.5 MO-'lane, 8 cm length gti. 1XTBE.5 V/cm. 1 h 5 0.5 pn/lane, 20 cm length pel, 1XTAE,8Wcm,3h Practical Part 3 Practical implementation of ELPHO 2. Electrophoresis of restrictive fragments after Taql cleavage Heterozygote Homozygote (cleaved) General Genetics Johann Gregor Mendel * 20. 7. 1822, Heizendorf t 6. 1. 1884, Brno Founder of genetics Discoverer of basic principles of inheritance • Principle of Fl generation uniformity • Principle of random segregation of genes • Principle of independent assortment of al into gametes leles Dominant Trait Flower Colour Plant Height Seed Color Seed Shape Pod Colour Pod Shape Flower Position * * w m \ \ Purple Tall Yellow Round Green Inflated (full) Axial Recessive Trait White Short Green Wrinkled Yellow Experiments on Plant Hybrids (1866) Constricted (flat) I Terminal Basic Concepts • Genetics • Science field examining heredity and variability of quantitative and qualitative traits of all living organisms • Gene • Basic unit of heredity (genetic information) • Sequence of DNA molecule carrying the information for production of protein or nucleic acid • Consists of exons and introns structural fUnCt'0nal 1^ E™ Intron E™ Intron E«on Intron E™ Intron E*™ RNA mRNA i..........J.....t............. Intron Exon Intron E*on Basic Concepts • Chromosome • Functional unit of hereditary record of genetic information in a cell • Cell nucleus —► 22 pairs of autosomes + 1 pair of gonosomes Locus • Position of a gene on a particular location on a specific chromosome Allele • Specific variant of the gene Chromosome Nucleus Chromatid Chromatid V/} f/y >/, Nucleosome Gene Solenoid loops Image adapted from: National Human Genome Research Institute. Basic Concepts • Genomics • Field of genetics trying to determine the whole genetic information of an organism and to interpret it in terms of life processes • Heterozygote • Two different variants (alleles) of a particular gene or its part • Homozygote • Two same variants (alleles) of a particular gene or its part Dominant homozygot Recessiv homozygot Dominant heterozygot B B b b B J b Basic Concepts Polymorphism • Existence of several (at least two) alleles for a specific gene, of which the least common one has population frequency > 1 % Mutation • Processes, during which changes in genotype occur due to different environmental factors • Less common allele has population frequency < 1 % DNA vs. RNA DNA molecule = deoxyribonucleic acid - Double helix - 2 strands in opposing directions - Polynucleotide chain • Nitrogenous bases ( X A, C, G) connected with hydrogen bonds • Phosphate group • Sugar - deoxyribose e, ö e o Om^P o o Base -nucleoside--nucleotide-- glycosidic bond RNA molecule = ribonucleic acid - One strand - Polynucleotide chain "vJL Ä • Nitrogenous bases ( U, A, C, G) connected with hydrogen bonds • Phosphate group ^ • Sugar - ribose - Types - mRNA, tRNA, rRNA ■nucleoside diphosphate-nucleoside triphosphate- Hue pur Adenine >~Y^*n Quinine H Cytoilne u Adenine >~Y^»h Guanine Of DNA DNA Deoxyribonucleic Acid RNA Ribonucleic Acid Nucelobases of RNA Central Dogma of Molecular Biology Nucleus Cytoplasm - ribosomes . Replication / (DNA synthesis) j / DNA ■■■■■■■■ ■■■■■■■■■■■■i ■■■■■■■■■■■■■■■■■■■■a Transcription (RNA synthesis) RNA ■■■■■■■■ Translation (protein synthesis) PROTEIN Amino acids DNA Replication = production of copies of DNA molecules providing genetic information transmission from parental to daughter cell • S-phase of cell cycle • semiconservative process - 1 new + 1 old strand Components required for replication • template - parental strand • primer - short oligonucleotide with free 3'OH end • enzymes • nucleotides DNA Replication Forming of the replication fork • helicase - allows separation of both molecules of the double-helix • SSB proteins - helps keep strands separated DNA primase - production of RNA primers Replication is started in specific locations -replication origins DNA polymerase - catalyzes elongation of the strand • sequence of the new strand according to the principle of complementarity of bases -adenine + thymine (2 hydrogen bonds) and cytosine + guanine (3 hydrogen bonds) • synthesis from 5'end to 3'end DNA polymerase Lagging stand DNA replication Original DNA I III 11/ Okazaki fragment RNA primer CI*™ Leading stand Topoisomerase Parent DNA DNA Replication Template strands are antiparallel - one strand is lagging • Leading strand - one RNA primer at the beginning, replication without interruption • Lagging strand - in direction 5'- 3' are discontinuously produced short Okazaki fragments (every from a new RNA primer), later connected by DNA ligase • RNA primers are removed by 5'-3' exonuclease activity and replaced by 3'-5' polymerase activity Transcription transcription of the information in a form of DNA sequence to RNA sequence cell nucleus template - DNA strand transcripts are from the template released as single strands DNA-dependent RNA polymerase • 3 types (similar structure, transcribing different types of genes) • RNA pol. I (genes coding rRNA) • RNA pol. II (genes coding hnRNA) • RNA pol. Ill (genes coding tRNA) • requires presence of transcriptional factors (separating DNA strands, placement of RNA polymerase to promoter and releasing from promoter) • Promoter = starting point on DNA - TATA box, CAT box • Terminator = ending point - AAAA G TG T GT GG GTT G TTA A G "GTATT > ) ) I I I > I I I I I I I I I I I I I I I I I I I I I I I I I i I I I I Sense Strand Post-transcriptional Modification Modification of primary transcripts: • addition of a cap to 5'end (helps controlling mRNA translation) Start of transcription f^POOjGCf Of r Gene: Intron 1 Intron 2 Exon 1 Obrázek 3 - Struktura tzv. RNA čepičky Exon 2 Exon 3 Transcription H2U H ooo ii ii ii -p-o-p-o-p-o I I o' 0' o 0 ov o=p-o 1 0 7-methylguanosine -O Primary transcript: | } Intron 1 Intron 2 Exonl \ / Exon 2 \/Exon 3 • connection of polyadenylic chain to 3'end • RNA splicing - cutting out of introns to form mature m R N A Mature transcnpt: Splicing Exon 1 Exon 2 Exon 3 Translation Translation of genetic information from mRNA to AA sequence in polypeptide (using genetic code) Occurs on ribosomes in cell cytoplasm • Phases - initiation, elongation, termination • Enzyme - Aminoacyl-tRNA synthetase • Initiation complex is formed at the 5'end of mRNA (cap), searching mRNA from 5'end, looking for initiation codon AUG • Termination of translation: UAA, UAG, UGA • Post-translational modifications-phosphorylation, glycosylation, methylation,.... Genetic Code • System for adding specific AAs to polypeptide chain according to mRNA sequence • Triplet = codon - defines AA or terminates translation • Every AA defined by one or several codons in mRNA • 64 possible triplets: 61 define AA, 3 terminate translation • Codons are recognized by complementary sequences in tRNA (anticodons), which carry specific AAs on 3' end • Insertion/deletion of one/two base pairs changes reading frame • (almost) universal, degenerate RNA codon table 2nd position 1st position u c A G 3rd position u Phe Phe Leu Leu Ser Ser Ser Ser Tyr Tyr stop stop Cys Cys stop Trp u c A G c Leu Leu Leu Leu Pro Pro Pro Pro His His Gin Gin Arg Arg Arg Arg U C A G A lie He He Met Thr Thr Thr Thr Asn Asn Lys Lys Ser Ser Arg Arg U C A G G Val Val Val Val Ala Ala Ala Ala Asp Asp Glu Glu Gly Gly Gly Gly U C A G Amino Acids Ala: Alanine Gin: Glutamine Leu: Leucine Ser: Serine Arg: Arginine Glu: Glutamic acid Lys: Lysine Thr: Threonine Asn: Asparagine Gly: Glycine Met: Methionine Trp: Tryptophane Asp:Aspartic acid His: Histidine Phe: Phenylalanine Tyr: Tyrosisne Cys:Cysteine He: Isoleucine Pro: Proline Val: Valine Genetic Code - Reading Frame Shift Normal open reading frame Amino acids I-^ / v ^- ' Met Thr Asp Gin Pro Gin Tyr Glu Leu Ala Phe Lys Ala Asp Ala Pro mRNA -I-1,1,1-1-1-1-1-1-1-1-1-1-1-1-L_^. AUGACGGAUCAGCCGCAAUACGAAUUGGCGUUUAAGGCGGAUGCGCCG ■ ^^^^ * /s/^^ ' Codons Sense strand (Coding strand)_ ATGACGGATCAGCCGCAATACGAATTGGCGTTTAAGGCGGATGCGCCG DNA..........................i.......i..........ill TACTGCCTAGTCGGCGTTATGCTTAACCGCAAATTC CGCCTACGCGGC , Antisense strand (Non-coding strand) Frameshift mutation - single nucleotide insertion open reading frame mRNA —I QValJ i GUU U A A GGCGGAUGCGCCG'' Codons Sense strand (Coding strand) Insertion ATGACGGATCAGCCGCA ATACGAATTGGCGTTTAAGGCGGATGCGCCG DNA.........I................I...................... TACTGCCTAGTCGGCGTrTATGCTTAACCGCAAATTCCGCCTACGCGGC , -KJ- Antisense strand (Non-coding strandf^ Cell Cycle M S = organized sequence of processes during which the cell doubles its content and subsequently divides into two daughter cells (both of them carry the same chromosomes) Aim: Reproduction of genetic material for the next generation of cells Cell Cycle high accuracy requirements • flawless replication • correct sequence of phases • mitosis before finished replication -> loss of genetic information in at least one cell • double replication before mitosis -> increased number of gene copies in a particular part of chromosome -> instability in gene expression, low viability • precise segregation of chromosomes „ • coordination with developmental programs \ — p checkpoints Cell Cycle - Interphase Interphase-Gl, G2, S • preparation for cell division, outer nuclear membrane connected to ER • unfavorable conditions • stopping in Gl/entering GO; cells do not grow, may stay suspended for several months/years GO-phase • most of cells in multicellular organisms (differentiated and specialized cells performing one function, do not divide) • after receiving pro-growth factor may enter back into the cell cycle Gl-phase • the longest and the most variable • the cell grows and doubles its organelles • checkpoint at the end of this phase: restriction point • the cell has abundant nutrients and growth factors, shows high metabolic activity -> passes the restriction point and enters the next phase • nutrient deficiency anti-proliferative signals -> slowing down of the phase progression/exiting of the cell cycle (entering GO) Cell Cycle - Mitosis nuclear division (mitosis) + subsequent cytoplasm division = cytokinesis Mitosis • division of somatic cells • product - two diploid cells with identical genetic information • prophase - spiralization of DNA strands, formation of the mitotic spindle • prometaphase - breakdown of the nuclear membrane • metaphase - formation of kinechotore on every centromere, connection of chromatids to spindle (equatorial plane) • anaphase - separation of chromatids to the opposing poles of the cell • telophase - finalization of chromatid separation, breakdown of spindle, recovery of the nuclear membrane Prophase Metaphase Anaphase Cytokinesis Mitosis vs. Meiosis J DMA REPLICATION paternal homolog maternal homolog DNA REPLICATION \ PAIRING OF DUPLICATED HOMOLOGOUS CHROMOSOMES BIVALENTS LINE UP ON THE SPINDLE DUPLICATED CHROMOSOMES LINE UP INDIVIDUALLY ON THE SPINDLE CELL DIVISION DIOIOIOMJIO Mitosis = 2 daughter cells with diploid number of chromosomes; 1 cycle of DNA replication, followed by separation of chromosomes and nuclear division (prophase —> prometaphase —> metaphase —> anaphase —> telophase) and subsequently the whole cell division (cytokinesis) Meiosis = 1 cycle of replication followed by 2 cycles of chromosome segregation and cell division, formation of haploid gametes first meiotic (reductional) division - separation of homologous chromosomes; meiotic crossing-over occurs here (gene recombination) - no two gametes are identical! separation disorders - e.g. trisomy. second meiotic division - separation of daughter chromatids -> 2 daughter cells with haploid number of chromosomes, formation of germ cells (sperm, egg), further swapping of genetic material by crossing-over gametes Meiosis formation of 4 haploid gametes (germ cells) genetic variability Heterotypic division • Prophase - 5 stages • Leptotene - spiralization • of chromosomes • Zygotene - formation of bivalents • Pachytene - crossing over = crossing of non-sister chromatids • Diplotene - gradual separation of homologous chromosomes • Diakinesis - dispersion of nuclear membrane, formation of spindle apparatus • Metaphase - formation of equatorial plane • Anaphase - division of 2n chromosomes, separation via spindle • Telophase - formation of nuclear membrane, breakdown of spindle Meiosis I interphase I Centrosomes (with centriole pairs) prophase I Chiasm at a metaphase I Microtubule attached to Metaphase kinetochore plate anaphase I Sister chromatids remain attached Nuclear Chromatin envelope Chromosomes duplicate Homologous chromosomes pair and exchange segments Synapsis - pairing of homologs to form tetrad Centromere (with kinetochore) Tetrads line up Homologous chromosomes separate Pairs of homologous chromosomes split up Meiosis Homeotypic division • Prophase - spiralization of chromosomes, formation of the spindle, dispersion of nuclear membrane • Metaphase - formation of equatorial plane, connection to spindle • Anaphase - separation of chromatids from divided chromosomes • Telophase - formation of nuclear membrane, breakdown of spindle, despiralization of chromosomes Meiosis I Meisois II _^_ telophase & prophase II metaphase II anaphase II telophase II chromosomes CflpyngfHOPeatson Edueallwv Inc.. pushing a* Bsctamm Cunurwifls. Mutation Mutations are genetic changes on the level of genetic material, which are manifested in the change of the primary structure of the nucleic acid, i.e. change in the sequence of nucleotides. They are related to changes of genotype, but do not have to manifest in phenotype. • Significance of mutations: • Positive - source of genetic variability, evolutionary significance • Negative - cumulation of damaged genes, rise of genetically determined diseases, tumors • Neutral • Classification of mutations: • spontaneous (errors in replication) x induced (mutagens) • gametic x somatic • dominant x recessive (1:100) • direct x reverse (mutated genotype reverses to the original genotype) • vital x lethal • nuclear x non-nuclear (mt, cp) • gene x chromosomal (structural CHA) x genomic (numerical CHA, aneuploidy, polyploidy) Gene Mutations • Gene (point) mutations • base substitution (transition, transversion) -standard allele -> mutant allele -> changed protein • deletion/insertion of bases (reading frame shift) • Consequences of point mutations: • mutations changing the meaning of a codon (different AA) • nonsense mutations (stop codon) • silent mutation (different codon, same AA) • Sickle cell anemia - AR inheritance; substitution CTC -> CAC in beta chain (substitution of valine for glutamic acid); resistance to malaria (Plasmodium infection) -heterozygous advantage (selective advantage compared to both homozygotes) (A) Normal SequencG (no mutation] (B) Insertion ("G" added) (C) Deletion ("A" removed) (D) Duplication ("C " repeated) (E) Inversion (" A" reversed) Types of genetic variants The gray cat ran down the hall, original The gray cat ran down the ball. Missense The gray green cat ran down the hall, insertion The gray_ran down the hall. Deletion 1 The gray cat cat ran down the hall. Duplication The gray. Nonsense Chromosomal Mutations Structural chromosomal aberrations are a product of one or more breaks in the DNA. * Classification: • balanced (same amount of genetic material) • translocation - e.g. Philadelphia chromosome t(9;22) in CML • inversion • insertion • unbalanced (portion of genetic material is missing or extra) • duplication • deletion • Cri du chat (deletion on short arm of chr. 5) • Prader-Willi syndrome (deletion of paternal chr. 15) • Angelman syndrome (deletion of maternal chr. 15) II 46,XX,del(5)(pl4.1) 11 • isochromosome • ring chromosome )l ii n ii h Ii ii its Genomic Mutations Karyotype from a female with Patau syndrome (47,XX,*13) • Aneuploidy of gonosomes • Turner syndrome 45, X • Klinefelter syndrome 47, XXY • XXX syndrome • XYY syndrome • Aneuploidy of autosomes • Down syndrome (21) • Edwards syndrome (18) • Patau syndrome (13) • Polyploidy • rare in vertebrates, more common in plants .... i 6 13 19 I! i) 3 4 V. 5 i! (I • • ■ . •1 -« ;; ■ • 7 8 9 10 11 i -■ : [( \ • )t •i 14 15 16 17 18 \/ -: » • - ■ ( \)i ) 20 21 22 V o< x v_y 12 )\)\ II 1 2 3 'Á )l H II if !l M 11 12 :s II 6 I 1 1 m ^14 II ii ii Normal karyotype iir in hi Triploidy — ii ní ii Trisomy ii i ii Monosomy Aneuploidy 17 11 21 22 x DNA Diagnostics Detection of presence of specific nucleic acid sequence • Identification of animal species • Paternity • Identification of individuals - forensics • DNA profile-SNPs Analysis of structure (sequence) of nucleic acid Determination of genotype • Detection of clinically significant mutations and polymorphisms • Hereditary diseases • Detection of oncogenes and suppressor genes in tumors Prenatal, preimplantation diagnostics Quantification of nucleic acid with specific sequence • Evaluation of intensity and changes in gene expression -tumors Quantification of proteins and types of their posttranslational modifications Autosomal recessive 3 3 Unaffected son Daughter carrier Son earner Affected daughter probata 1:4 2:4 2:4 1:4 *_> Recommended Literature for Self-study ulij jĽm Thank you for you attention