5 - Methods.pptx.pdf

The Techniques of Molecular Genetics Dr. Madona Akhobadze 2024  Tools of Human Molecular Genetics. Analysis of Individual DNA and RNA Sequences. Methods of Nucleic Acid Analysis. Restriction Endonucleases. T...

The Techniques of Molecular Genetics Dr. Madona Akhobadze 2024  Tools of Human Molecular Genetics. Analysis of Individual DNA and RNA Sequences. Methods of Nucleic Acid Analysis. Restriction Endonucleases. The Production of Recombinant DNA Molecules In Vitro. Construction and Screening of DNA Libraries Reading: Ch. 14 - Principles of Genetics, by Snustad & Simmons Ch. 4 - Thompson & Thompson Genetics in Medicine, Robert L. Nussbaum, Roderick R. McInnes HYPOPITUITARY DWARFISM year 1972 molecular structure and function of the human growth hormone (hGH) gene  IMAGINE THAT YOU ARE A GENETICIST… GENE CLONING HUMAN INSULIN, WHICH WAS THE FIRST SUCH PRODUCT, WAS APPROVED IN 1982 1985, HGH PRODUCED IN E. COLI Greek: klon, meaning twig One product of any cloning experiment is a clone, group of identical cells or organisms  GENE CLONING CLONES OF IDENTICAL FROGS - JOHN GURDON SHEEP NAMED DOLLY WAS CLONED IN SCOTLAND IN 1997 STANLEY COHEN, HERBERT BOYER, AND THEIR COLLEAGUES PERFORMED THE FIRST CLONING EXPERIMENT IN 1973 THE ROLE OF RESTRICTION ENDONUCLEASES STEWART LINN AND WERNER ARBER DISCOVERED RESTRICTION ENDONUCLEASES IN E. COLI IN THE LATE 1960 GREEK: ENDO, MEANING WITHIN HAEMOPHILUS INFLUENZAE STRAIN RD, DISCOVERED BY HAMILTON SMITH HINDII (PRONOUNCED HIN-DEE-TWO) HINDII RECOGNIZES THIS SEQUENCE: ↓ GTPYPUAC CAPUPYTG ↑  ECORI (PRONOUNCED EEKO R-1 OR ECHO R-1) ↓ 59---GAATTC---39 ---G39 59AATTC--- 39---CTTAAG---59 ---CTTAA59 39G--- ↑ PSTI SMAI  AND MADAM DNA PALINDROMES NAPOLEON’S LAMENT: “ABLE WAS I ERE I SAW ELBA,” A WART REMEDY: “STRAW? NO, TOO STUPID A FAD; I PUT SOOT ON WARTS,” STATEMENT OF PREFERENCE IN ITALIAN FOOD: “GO HANG A SALAMI! I’M A LASAGNA HOG.”  RESTRICTION–MODIFICATION SYSTEM R-M SYSTEM HALF-METHYLATION (HEMIMETHYLATION) IS ENOUGH TO PROTECT THE DNA DUPLEX AGAINST CLEAVAGE PLASMIDS, SMALL, CIRCULAR DNAS THAT ARE INDEPENDENT OF THE HOST CHROMOSOME PSC101 - RESISTANCE TO THE ANTIBIOTIC TETRACYCLINE RSF1010 - RESISTANCE TO STREPTOMYCIN AND SULFONAMIDE  ECORI CUT THESE CIRCULAR DNAS, IT CONVERTED THEM TO LINEAR MOLECULES DNA LIGASE COMPLETED THE TASK OF JOINING THE TWO DNAS COVALENTLY RECOMBINANT DNA  SUMMARY RESTRICTION ENDONUCLEASES RECOGNIZE SPECIFIC SEQUENCES IN DNA MOLECULES AND MAKE CUTS IN BOTH STRANDS. THIS ALLOWS VERY SPECIFIC CUTTING OF DNAS. ALSO, BECAUSE THE CUTS IN THE TWO STRANDS ARE FREQUENTLY STAGGERED, RESTRICTION ENZYMES CAN CREATE STICKY ENDS THAT HELP LINK TOGETHER TWO DNAS TO FORM A RECOMBINANT DNA IN VITRO  Vectors serve as carriers to allow replication of recombinant DNAs an origin of replication plasmids and phages  TRANSFORMATION INCUBATE THE CELLS IN A CONCENTRATED CALCIUM SALT SOLUTION TO MAKE THEIR MEMBRANES LEAKY THEN MIX THESE PERMEABLE CELLS WITH THE DNA TO ALLOW THE DNA ENTRANCE TO THE LEAKY CELLS. USE HIGH VOLTAGE TO DRIVE THE DNA INTO CELLS—A PROCESS CALLED ELECTROPORATION.  PHAGES AS VECTORS NATURAL ADVANTAGE OVER PLASMIDS NOT COLONIES OF CELLS, BUT PLAQUES GENETICALLY IDENTICAL—A CLONE CHARON PHAGES REPLACEMENT VECTORS CHARON 4 CAN ACCEPT UP TO ABOUT 20 KB OF DNA CONSTRUCTING GENOMIC LIBRARIES  IF THE ENZYME CUTS GENOMIC LIBRARIES ONLY ABOUT EVERY FOURTH OR FIFTH SITE 16–20 KB NO CATALOG EXISTS AN IDEAL PROBE WOULD BE A LABELED NUCLEIC ACID WHOSE SEQUENCE MATCHES THAT OF THE GENE OF INTEREST PLAQUE HYBRIDIZATION  COSMIDS DESIGNED ESPECIALLY FOR CLONING LARGE DNA FRAGMENTS BEHAVE BOTH AS PLASMIDS AND AS PHAGES ROOM FOR LARGE INSERTS (40–50 KB)  M13 PHAGE VECTORS FILAMENTOUS (LONG, THIN, FILAMENT-LIKE) PHAGE  WHAT IS THE ADVANTAGE OF THE M13 VECTORS? SINGLE-STRANDED DNA CAN BE RECOVERED IN SINGLE-STRANDED FORM — A COMPLEMENTARY DNA LIBRARY—  PHAGEMIDS PRODUCE SINGLE-STRANDED DNA HAVE CHARACTERISTICS OF BOTH PHAGES AND PLASMIDS  Chromosome Identification THERE ARE THREE COMMONLY USED STAINING METHODS THAT CAN DISTINGUISH AMONG HUMAN CHROMOSOMES GIEMSA BANDING (G BANDING) - THE MOST COMMON METHOD USED IN CLINICAL LABORATORIES  Chromosome Identification Q BANDING - REQUIRES STAINING WITH QUINACRINE MUSTARD OR RELATED COMPOUNDS AND EXAMINATION BY FLUORESCENCE MICROSCOPY  Chromosome Identification R BANDING – HEATING BEFORE STAINING, THE RESULTING DARK AND LIGHT BANDS ARE THE REVERSE OF THOSE PRODUCED BY G OR Q BANDING AND ARE ACCORDINGLY REFERRED TO AS R BANDS  Chromosome Identification C BANDING – STAINING THE CENTROMERIC REGION OF EACH CHROMOSOME AND OTHER REGIONS CONTAINING CONSTITUTIVE HETEROCHROMATIN  Special Cytological Procedures C BANDING – STAINING THE CENTROMERIC REGION OF EACH CHROMOSOME AND OTHER REGIONS CONTAINING CONSTITUTIVE HETEROCHROMATIN HIGH-RESOLUTION BANDING - CALLED PROMETAPHASE BANDING) IS ACHIEVED THROUGH G-BANDING OR R-BANDING TECHNIQUES TO STAIN CHROMOSOMES FRAGILE SITES - NON-STAINING GAPS THAT ARE OCCASIONALLY OBSERVED AT CHARACTERISTIC SITES ON SEVERAL CHROMOSOMES  FLUORESCENCE IN SITU HYBRIDIZATION FISH - TECHNIQUE TO EXAMINE THE PRESENCE OR ABSENCE OF A PARTICULAR DNA SEQUENCE OR TO EVALUATE THE NUMBER OR ORGANIZATION OF A CHROMOSOME OR CHROMOSOMAL REGION IDENTIFY PARTICULAR CHROMOSOMAL REARRANGEMENTS OR TO RAPIDLY DIAGNOSE THE EXISTENCE OF AN ABNORMAL CHROMOSOME NUMBER IN CLINICAL MATERIAL  THESE PROBES “PAINT” THE TARGET CHROMOSOME COMPARISON OF CELLS IN METAPHASE AND INTERPHASE VISUALLY DOCUMENTS THE DYNAMIC NATURE OF CHROMOSOME CONDENSATION AND DECONDENSATION THROUGHOUT THE CELL CYCLE  CHROMOSOME AND GENOME ANALYSIS BY USE OF MICROARRAYS COMPARATIVE GENOME HYBRIDIZATION – CGH COMPLEMENTS CONVENTIONAL KARYOTYPING AND HAS THE POTENTIAL TO PROVIDE A MUCH MORE SENSITIVE, HIGH-RESOLUTION ASSESSMENT OF THE GENOME REVEAL VARIANTS, IN PARTICULAR SMALL CHANGES IN COPY NUMBER BETWEEN SAMPLES, THAT ARE OF UNCERTAIN CLINICAL SIGNIFICANCE.  PCR. Real Time PCR. Nucleotide Sequences of Genes and Chromosomes Reading: Ch. 14 - Principles of Genetics, by Snustad & Simmons Ch. 4 - Molecular Biology, Weaver Robert J. McGraw Hill Kary Mullis and his colleagues in the1980s uses the enzyme DNA polymerase to make a copy of a selected region of DNA JURASSIC PARK SUMMARY PCR amplifies a region of DNA between two predetermined sites. Oligonucleotides complementary to these sites serve as primers for synthesis of copies of the DNA between the sites. Each cycle of PCR doubles the number of copies of the amplified DNA until a large quantity has been made.  ANALYSIS OF RNAS BY REVERSE TRANSCRIPTASE-PCR (RT-PCR) THE ENZYME REVERSE TRANSCRIPTASE CATALYZES THE SYNTHESIS OF DNA STRANDS THAT ARE COMPLEMENTARY TO RNA TEMPLATES. IT CAN BE USED IN VITRO TO SYNTHESIZE DNAS THAT ARE COMPLEMENTARY TO RNA TEMPLATE STRANDS USING REVERSE TRANSCRIPTASE PCR (RT-PCR) IN CDNA CLONING clone a cDNA from just one mRNA starts with an mRNA instead of a double-stranded DNA RNA→DNA reverse transcribes the mRNA to make a single-stranded DNA, then uses a forward primer to convert the single-stranded DNA to double-stranded.  THE RESULTING DNA STRANDS CAN THEN BE CONVERTED TO DOUBLE-STRANDED DNA BY SEVERAL DIFFERENT PROCEDURES, INCLUDING THE USE OF A SECOND PRIMER AND THE HEAT-STABLE TAQ DNA POLYMERASE. THE RESULTING DNA MOLECULES CAN THEN BE AMPLIFIED BY STANDARD PCR THE FIRST STRAND OF DNA, OFTEN CALLED A C-DNA BECAUSE IT IS COMPLEMENTARY TO THE M-RNA UNDER STUDY, CAN BE SYNTHESIZED BY USING AN OLIGO (DT) PRIMER THAT WILL ANNEAL TO THE 3-POLY(A) TAILS OF ALL M-RNAS, OR BY USING GENE-SPECIFIC PRIMERS GENE-SPECIFIC OLIGONUCLEOTIDE PRIMERS ARE USUALLY CHOSEN TO ANNEAL TO SEQUENCES IN THE 3-NONCODING REGIONS OF THE M-RNAS.  THE PRODUCTS OF THESE AMPLIFICATIONS ARE ANALYZED BY GEL ELECTROPHORESIS. WHEREVER A PRODUCT APPEARS IN THE GEL, THE INVESTIGATOR KNOWS THAT THE SAMPLE FROM WHICH IT WAS GENERATED CONTAINED THE M-RNA UNDER STUDY. THIS PROCEDURE IS THEREFORE A QUICK AND EASY WAY OF ASCERTAINING WHETHER OR NOT A PARTICULAR GENE IS BEING TRANSCRIBED. MANY MODIFICATIONS OF THE RT-PCR PROCEDURE HAVE BEEN DEVELOPED, WITH A MAJOR EMPHASIS ON MAKING IT MORE QUANTITATIVE. FOR EXAMPLE, KNOWN AMOUNTS OF THE RNA UNDER STUDY CAN BE ANALYZED TO DETERMINE THE RELATIONSHIP BETWEEN RNA INPUT AND DNA OUTPUT. BY KNOWING THIS RELATIONSHIP, AN INVESTIGATOR CAN USE THE QUANTITY OF DNA GENERATED BY AN EXPERIMENTAL SAMPLE TO EXTRAPOLATE BACK TO THE AMOUNT OF RNA THAT WAS INITIALLY PRESENT IN THAT SAMPLE  SUMMARY RT-PCR can be used to generate a cDNA from a single type of mRNA, but the sequence of the mRNA must be known so the primer for the PCR step can be designed. Restriction site sequences can be placed on the PCR primers, so these sites appear at the ends of the cDNA. This makes it easy to cleave them and then to ligate the cDNA into a vector REAL-TIME PCR quantifying the amplification of a DNA as it occurs—that is, in real time reporter probe The forward and reverse primers (purple) are annealed to the two separated DNA strands (blue), and a reporter probe (red) is annealed to the top DNA strand. The reporter probe has a fluorescent tag (gray) at its 59-end and a fluorescence quenching tag (brown) at its 39-end. DNA polymerase has extended the primers, with the new DNA depicted in green. To make way for replicating the top strand, the DNA polymerase has also degraded part of the reporter probe. This separates the fluorescent tag from the quenching tag, and allows the fluorescent tag to exhibit its normal fluorescence (yellow). The more DNA strands are replicated, the more fluorescence will be observed. SUMMARY Real-time PCR keeps track of the progress of PCR by monitoring the degradation of a reporter probe hybridized to the strand complementary to the forward primer. As this probe is degraded, a fluorescent tag is separated from a quenching tag, so fluorescence increases, and this increase can be measured in real time in a fluorimeter გმადლობთ , ყურადღებისთვის !!!