Methods of molecular genetics I-II.

1. Methods of molecular genetics I-II. (EPh 2017)

2. Study of DNA

3. The basic principles - the most important molecular genetic methods can be classified into four types • "Cut-and-paste” • Hybridization • Amplify • Visualize • Restriction endonucleases + ligases • E.g. FISH • PCR • Electrophoresis

4. Sequence specific restriction enzymes cut the dsDNA • restriction site (palindromic sequence) • Bacterial endonuclease protects the bacterium from bacteriophage by cut up the foreign DNA without impair a DNA of the bacterium • "Cut-and-paste”

5. Restriction ezymes • "Cut-and-paste”

6. Recombinant DNA • "Cut-and-paste”

7. Insert + vector (plasmid, virus, eukaryotic arteficial chromosome) = Recombinant DNA

8. Application of recombinant DNA technology • Generation of transgenic organisms, cells, pathogens etc. for medical research KO (knock out) – gene is inactive Transgenic – gene is overexpressed

9. Gene expression for therapeutic purpose • Typical recombinant drugs: • protein-hormones • vaccine-antigens • antibodies • cytokines • Growth factors • Coagulation factors • Bioreactor producing recombinant insulin

10. Hybridization and Fluorescence in situ hybridyzation (FISH) ds oligonucleotide Indirect Direct Ag Fluorophore Probe = labeled oligonucleotide labeling denaturation hybridization Ag Ab- fluorophore Detection in fluorescent microscope Hybridization

11. TELOMERE DETECTION BY in situ HYBRIDIZATION 11 Hybridization

12. NOR = Nucleolar organizer region = contains repetitive sequences 12 Hybridization

13. Fluorescence in situ hybridyzation (FISH)I. Y chromosome specific probe Hybridization

14. FISH II. Application of both X and 21 chromosome specific probes Hybridization

15. TRISOMY OF CHROMOSOME 21 (interphase FISH) 15 Hybridization

16. Szűrés élőben előforduló lehetséges számbeli kromoszóma rendellenességekre Prenatal diagnostics 16 www.lim.cz/files/File/lucia/leaflets/PGD_cytogen.pdf Hybridization

17. Spectral karyotyping (SKY) or multicolor FISH For each chromosome different dye labelled mixture of specific probes are used. Hybridization

18. Normal sample Tumor cell Hybridization

19. COMBINATIONS OF LABELLING FLUOROCHROMES 19 Hybridization

20. CHROMOSOME TERRITORIES (HUMAN) 20 Hybridization

21. Polymerase chain reaction-PCR generation of thousands to millions of copies (amplification) of a or a few copies of DNA sequences in a short time (in vitro DNA synthesis) The thermostable DNA polymerase (Taq polymerase) enzyme of Thermus aquaticus (lives in extremely high temperature) is needed Typically amplify DNA fragments between 0.1 and 5kb Errors: If the DNA is not clean enough, contamination will be amplifyied as well! Necessary: pair template 1 2 3 three temperature dependent steps 21

22. Polymerase chain reaction (PCR) I. Amplify

23. PCR II. Amplify

24. Electrophoresis = Separation of DNA fragments I. Vizualize

25. Electrophoresis II. Visualization of DNA fragments (Stained by eg. Ethidium bromide + UV light - not specific) stained gels Photo of a stained gel

26. Southern blot I. Digestion with restriction enzyme

27. Southern blot II. Gel electrophoresis

28. Southern blot III. Blotting (transfer from gel onto nitrocellulose filter)

29. Southern blot IV. Hybridization • Nucleic acid probes: • single stranded nucleic acid • sequence specific (not at • polymorph region) • labelled by isotope • (or fluorescence dye) Can be detected by autoradiography

30. Southern blot V.Autoradiography • Specific nucleic acid used for hybridization is labelled by  particles (electrons) emitting radioactive isotope (most frequently 32P) •  particles reduce Ag+ to Ag(not visible) in Xray film put on the nitrocellulose membrane • Ag is visualized by phototechnical steps: development and fixation • result: black lanes show the DNA fragments that hybridized with the probe

31. Southern blot VI. Non-specific specific

32. Southern blot V. (semiquantitative) DNA copy number

33. Application of molecular genetic technique • Analysis of geneticbackgroundofpatient = Genotyping • Recognition site of restrictionenzymesor the distancebetweenthemmaychangeduetopolymorphisms and mutations (SNP (SNV), STR or VNTR) • Size of fragmentsmay be checkedbyelectrophoresis, differentcleavageresultsdifferentfragmentpattern = RFLP (restrictionfragmentlengthpolymorphism)

34. DNA polymorphisms I SNP (single nucleotid polymorphism) SNV (single nucleotid variant) (variant 1) (Variant 2) -1 -2 2 DNA variants (alleles); 3 geno(pheno)types (of diploid organism)

35. DNA polymorphisms II SNV/Restriction fragment length polymorphism (RFLP)/ Southern blot

36. DNA polymorphisms III SNV/Restriction fragment length polymorphism Southern blot probe Red arrows: restriction sites

37. DNA polymorphisms IV./ Variable number of tandem repeat (VNTR) Variant 3....AGTC(CGAA)14CGCT…. Variant 4....AGTC(CGAA)18 CGCT…. Variant 5....AGTC(CGAA)20 CGCT…. Etc. Many DNA variants(alleles); much more geno(pheno)types

38. DNA polymorphisms V. /VNTR/RFLP/Southern blot DNA sequencevariants DNA fingerprint of 3 persons (diploid!)

39. DNA fingerprint DNS profile: genotype for all chromosome markers of an individual In forensic studies 11-13 chromosome markers are investigated Determination of sex: by the help of amelogenin polymorphism amelogenin gene is found on both X and Y, but the Y chromosome gene contains six base pairs which are missing from X chromosome gene Combined DNA Index System (CODIS) is a DNA database funded by the United States Federal Bureau of Investigation (FBI) 39

40. DNA analysis I. (based on VNTR) Who is the offender?

41. DNA analysis II. Who is the offender?

42. DNA analysis III. (based on VNTR) Who is the offender? Who is the father?

43. Use of PCR in genetic tests I-II.: PCR-RFLP and PCR-VNTR • The DNA region is amplified with specific primers in PCR • Digestion of DNA with restriction enzymes (for PCR-RFLP) • Gel electrophoresis • Staining with DNA-binding dyes, eg. Ethidium bromide • Evaluation in UV light PCR-VNTR PCR-RFLP N M H A1 A2 a (=b+c) b c PCR-basedproof forward and reverse primers

44. PCR – RFLP (SNV) * Polymorph site 1. PCR product: 450 bp 450 bp 282 bp 168 bp 2. Cleavage if a polymorph site is present cleaved PCR products: 282 bp 168 bp Marker 1 2

45. (cystic fibrosis)

46. PCR – RFLP (VNTR)

47. PCR –RFLP(VNTR)in forensic study

48. Multiplex PCR (usage in diagnosis) 401bp 347bp The upper band (401bp) represents WSSV (White spot syndrome virus). The lower band (347bp) represents IHHNV( Infectious Hypodermal Hepatopoietic Necrosis virus). Samples with two bands indicate dual infections. Samples withno bands indicate no infection.

49. Sequencing • Determination of the nucleotide sequence of DNA fragments • Maxam-Gilber and Sanger methods • Sanger method (chain termination) is widespread • Automation with labelled di-deoxynucleotides and capillary electrophoresis

50. Detection of point mutations: sequencingSanger method • Chain termination synthesis • PCR reaction with dNTP and ddNTP (dideoxyribonucleotides) • Because of the absence of 3'OH, the synthesis ends when ddNTP is incorporated • Shorter and longer fragments arise • Capillary electrophoresis: all four ddNTPs fluorescently labeled (four colors) • Laser reading: colored ladder peaks  base sequence