Research techniques in genetics. Medical genetics, 2017.

Transcription

1 Research techniques in genetics Medical genetics, 2017.

2 Techniques in Genetics Cloning (genetic recombination or engineering ) Genome editing tools: - Production of Knock-out and transgenic mice - CRISPR Cas9 (2014.) - TALEN (2010.); zinc finger nucleases (2000.) Stem cell production

3 Cloning (Genetic engineering)

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5 Cloning Natural or artificial development of two or more genetically identical cells or organisms Asexual propagation of plants and bacteria In molecular genetics creating recombinant genetic material by inserting foreign (e.g. human) DNA into plasmid DNA, which serves as a vector Such plasmids can be propagated in a large number of geneticaly identical copies

6 Plasmids In research used as vectors for gene transfer: Isolated segments of bacterial or viral DNA, capable of replicating independently of chromosomal DNA Double stranded and circular One bacterial cell can have up to a 1000 copies

7 Plasmid as a vector (multiple cloning site)

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9 Restriction endonucleases (multiple cloning site) (PCR product) Restriction endonucleases DNA ligase Recombinate plasmid vector

10 Cutting with EcoR1

11 Cloning into a plasmid vector Insert DNA fragments

12 The usage of recombinant plasmid: After successful cloning, recombinant plasmids have to be propagated in large number of copies and purified: 1. Transformation and selection (DH5alfa or BL21 E. coli) 2. Propagation growing in selective bacterial culture media 3. Purification isolation of bacterial plasmid DNA from culture How it can be used? 1. Introduction of genes into cells and organisms (transfection) 2. Production of recombinant proteins 3. Creating a Knock-out mouse

13 Usage 3: Knock-out mice (2007. Nobel prize in Physiology) Production of knock-out mice enabled us to study the influence of particular gene product on growth, developmen and function Procedure includes selective inactivation of certain gene in a faze of blastocist Today all genes of a mouse genome ( gene library ) are being tested through this technique Mouse genome will have a: Resistency towards one of the antibiotics (necessery for the process of cell selection) Gene for making a fenotipe for changed genotype easily detectable (cross-breading of white and black mouse)

14 TK- tymidin kinase NEO- neomycine cassette G418 - geneticine Making a knockout mouse: Stage 1, creating stem cells with an interrupted gene 8. Sellect with G418 and gangcyclovir

15 Making a knockout mouse: Stage 2, placing the errupted gene in the animal

16 CRISPR Cas9 Clustered Regularly Interspaced Short Palindromic Repeats CRISPR a prokaryotic immune system that confers resistance to foreign genetic elements (adoptive immunity) Requires Cas9 (enzyme nuclease) and guide RNA RNA guides Cas9, which cuts both DNA strands. Cuts are repaired with introduced mutations Alows editing parts of the genome by removing, adding or altering sections of the DNA sequence currently the simplest, most versatile and precise method of genetic manipulation

17 Human genome

18 Human genome 23 pairs of chromosomes (2n=46) one pair of sex chromosomes (XX ili XY) Chromosomes can be identified during cell division

19 Mitosis Mitosis = somatic cell (and sex cell!) division

20 Meiosis Meiosis = final phase in sex cell division gamete formation first meiotic division = reduction division haploid number of chromosomes Essencial for homologous recombination second meiotic division - simmilar to mitosis

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22 DNA based analysis techniques PCR, RT-PCR Restriction digestion (RFLP) Southern blot Sequencing: - Sanger method (dideoxy) - next generation sequencing, pyrosequencing Quantitative fluorescent PCR Multiple ligation dependant probe amplification (MLPA) USED for: SNP detection, mutation analysis, submicroscopic deletions, duplications,

23 Chromosome based analysis techniques Karyotype (G, R, C, etc. banding) Flow cytometry (EB stain) FISH Array comparative genomic hybridisation (Array CGH) USED for: deletions, aneuploidy, translocations, satelite polymorphism, fragile sites, copy number variations,

24 Karyogram (karyotype) Limphocytes incubated for 2-3 days or bone marrow cells within 4-24 hours

25 Cytogenetics karyotype analysis

26 PCR Kary Mullis (Nobel-chem:1993.)

27 PCR 40 cycle a trillion copies! (10 12 )

28 RT-PCR

29 RFLP

30 Sanger sequencing method

31 Frederic Sanger DNA Sequencing (Nobel chem: 1958 and 1980.)

32 DNA sequencing

33 Flourescente In Situ hybridisation- FISH Fluorescence marked oligonucleotydes ( probes ) specific for a chromosomal region (centromere, telomere, microdeletion specific, translocation specific, ) Hybridisation of probes to cromosome during renaturation (cooling) Comparative genomic hybridisation classical or microarray

34 Array CGH (Microarray analysis) A great number of oligonucleotides (probes) imobilised on a microchip as tiny dots (up to several thousands), in predesigned order Tested DNA (or cdna) is coloured green, and a control DNA is coloured red (egz: tumor vs. healthy control) After mixing, DNA is applied on a chip and hybridised with probes Detection of colour and intensity is used for DNA genotyping (copy number variation, mutations) or detection of difference in RNA expression, SNP detection, etc. (by software analysis)

35 Microarray

36 acgh results

37 Quantitative fluorescente PCR (For t hedetection of aneuploidy (trisomy or monosomy).

38 Multiplex ligationdependent probe amplification - MLPA Used for a detection of deletions or insertions on a specific location of chromosome (usualy for cancers mutations)

39 Multiplex ligationdependent probe amplification - MLPA

40 DNA fingerprinting analysis of minisatellite repeats Minisatellite: variable number of tandem repeats of bp, repeated 5-50 times, on thousands of locations in the human genome Microsatellite: short tandem repeats of 2-5 bp, repeated 5-50 times, on thousands of locations in the human genome

41 Southern blotting

42 Glossary A Haplotype - a combination of alleles at adjacent locations on the chromosome that are transmitted together (inhereted from a single parent) HapMap Project is an organization that aims to develop a haplotype map of the human genome, which will describe the common patterns of human genetic variation Genome-wide association study (GWA) - examination of many common genetic variants in different individuals to see if any variant is associated with a trait. GWAS typically focus on associations between single-nucleotide polymorphisms (SNPs) and traits like major diseases. Genetic linkage - the tendency of genes that are located proximal to each other on a chromosome to be inherited together during meiosis.

43 Candidate gene discovery path: 1. part

44 2. part