Molecular Genetics Techniques II 2024 PDF

The Techniques of Molecular Genetics II Dr. Madona Akhobadze 2024  The Molecular Analysis of DNA, RNA, and Protein. Analysis of RNAs by Reverse Transcriptase-PCR (RT-PCR) Reading: Ch. 14 - Principle...

The Techniques of Molecular Genetics II Dr. Madona Akhobadze 2024  The Molecular Analysis of DNA, RNA, and Protein. Analysis of RNAs by Reverse Transcriptase-PCR (RT-PCR) Reading: Ch. 14 - Principles of Genetics, by Snustad & Simmons Ch. 4 - Thompson & Thompson Genetics in Medicine, Robert L. Nussbaum,Roderick R. McInnes IMAGINE THAT YOU ARE A GENETICIST… Dolly, the first cloned mammal Finn Dorset ewe Blackface ewe  CLINICAL CYTOGENETICS IS THE STUDY OF CHROMOSOMES, THEIR STRUCTURE AND THEIR INHERITANCE, AS APPLIED TO THE PRACTICE OF MEDICAL GENETICS  CHROMOSOME ABNORMALITIES—MICROSCOPICALLY VISIBLE CHANGES IN THE NUMBER OR STRUCTURE OF CHROMOSOMES—COULD ACCOUNT FOR A NUMBER OF CLINICAL CONDITIONS THAT ARE THUS REFERRED TO AS CHROMOSOME DISORDERS  CHROMOSOME DISORDERS MAJOR CATEGORY OF GENETIC DISEASE ACCOUNT FOR A LARGE PROPORTION OF ALL REPRODUCTIVE WASTAGE, CONGENITAL MALFORMATIONS, AND MENTAL RETARDATION AND PLAY AN IMPORTANT ROLE IN THE PATHOGENESIS OF MALIGNANT DISEASE  INTRODUCTION TO CYTOGENETICS TO PREPARE A SHORT-TERM CULTURE THAT IS SUITABLE FOR CYTOGENETIC ANALYSIS OF THESE CELLS, SAMPLE OF PERIPHERAL BLOOD IS OBTAINED, USUALLY BY VENOPUNCTURE AND MIXED WITH HEPARIN TO PREVENT CLOTTING THE WHITE BLOOD CELLS ARE COLLECTED, PLACED IN TISSUE CULTURE MEDIUM, AND STIMULATED TO DIVIDE  INTRODUCTION TO CYTOGENETICS AFTER A FEW DAYS, THE DIVIDING CELLS ARE ARRESTED IN METAPHASE WITH CHEMICALS THAT INHIBIT THE MITOTIC SPINDLE, COLLECTED, AND TREATED WITH A HYPOTONIC SOLUTION TO RELEASE THE CHROMOSOMES CHROMOSOMES ARE THEN FIXED, SPREAD ON SLIDES, AND STAINED BY ONE OF SEVERAL TECHNIQUES, DEPENDING ON THE PARTICULAR DIAGNOSTIC PROCEDURE BEING PERFORMED. THEY ARE THEN READY FOR ANALYSIS.  MOLECULAR KARYOTYPING THE APPLICATION OF GENOMIC TECHNIQUES TO ASSESS THE INTEGRITY AND DOSAGE OF THE KARYOTYPE GENOME-WIDE THE DETERMINATION OF WHAT APPROACHES ARE MOST APPROPRIATE FOR PARTICULAR DIAGNOSTIC OR RESEARCH PURPOSES IS A RAPIDLY EVOLVING AREA, AS THE RESOLUTION, SENSITIVITY, AND EASE OF CHROMOSOME AND GENOME ANALYSIS INCREASE  CLINICAL INDICATIONS FOR CHROMOSOME ANALYSIS PROBLEMS OF EARLY GROWTH AND DEVELOPMENT STILLBIRTH AND NEONATAL DEATH FERTILITY PROBLEMS FAMILY HISTORY NEOPLASIA PREGNANCY IN A WOMAN OF ADVANCED AGE  SKIN BIOPSY, A MINOR SURGICAL PROCEDURE, CAN PROVIDE SAMPLES OF TISSUE THAT IN CULTURE PRODUCE FIBROBLASTS WHITE BLOOD CELLS CAN ALSO BE TRANSFORMED IN CULTURE TO FORM LYMPHOBLASTOID CELL LINES THAT ARE POTENTIALLY IMMORTAL  BONE MARROW CAN BE OBTAINED ONLY BY THE RELATIVELY INVASIVE PROCEDURE OF MARROW BIOPSY FETAL CELLS DERIVED FROM AMNIOTIC FLUID (AMNIOCYTES) OR OBTAINED BY CHORIONIC VILLUS BIOPSY  THE DEVELOPMENT OF RECOMBINANT DNA TECHNIQUES HAS SPAWNED MANY NEW APPROACHES TO THE ANALYSIS OF GENES AND GENE PRODUCTS GENETICISTS CAN ISOLATE AND CHARACTERIZE ESSENTIALLY ANY GENE FROM ANY ORGANISM; HOWEVER, THE ISOLATION OF GENES FROM LARGE EUKARYOTIC GENOMES IS SOMETIMES A LONG AND LABORIOUS PROCESS  ONCE A GENE HAS BEEN CLONED… ITS EXPRESSION CAN BE INVESTIGATED IN EVEN THE MOST COMPLEX ORGANISMS SUCH AS HUMANS. LET’S CONSIDER SOME OF THE MOST IMPORTANT METHODS USED TO INVESTIGATE THE STRUCTURE OF GENES (DNA), THEIR TRANSCRIPTS (RNA), AND THEIR FINAL PRODUCTS (USUALLY PROTEINS)  TO ANSWER QUESTIONS LIKE… IS A PARTICULAR GENE EXPRESSED IN THE KIDNEY, THE LIVER, BONE CELLS, HAIR FOLLICLES ERYTHROCYTES, OR LYMPHOCYTES? IS THIS GENE EXPRESSED THROUGHOUT THE DEVELOPMENT OF THE ORGANISM OR ONLY DURING CERTAIN STAGES OF DEVELOPMENT? IS A MUTANT ALLELE OF THIS GENE SIMILARLY EXPRESSED, SPATIALLY AND TEMPORALLY, DURING DEVELOPMENT? OR DOES THE MUTANT ALLELE HAVE AN ALTERED PATTERN OF EXPRESSION? IF THE LATTER, IS THIS ALTERED PATTERN OF EXPRESSION RESPONSIBLE FOR AN INHERITED SYNDROME OR DISEASE?  ANALYSIS OF DNAS BY SOUTHERN BLOT HYBRIDIZATIONS GEL ELECTROPHORESIS IS A POWERFUL TOOL FOR THE SEPARATION OF MACROMOLECULES WITH DIFFERENT SIZES AND CHARGES. DNA MOLECULES HAVE AN ESSENTIALLY CONSTANT CHARGE PER UNIT MASS; THEY SEPARATE IN AGAROSE AND ACRYLAMIDE GELS ALMOST ENTIRELY ON THE BASIS OF SIZE OR CONFORMATION.  AGAROSE OR ACRYLAMIDE GELS ACT AS MOLECULAR SIEVES, RETARDING THE PASSAGE OF LARGE MOLECULES MORE THAN SMALL MOLECULES. AGAROSE GELS ARE BETTER SIEVES FOR LARGE MOLECULES (LARGER THAN A FEW HUNDRED NUCLEOTIDES); ACRYLAMIDE GELS ARE BETTER FOR SEPARATING SMALL DNA MOLECULES  THE PROCEDURES USED TO SEPARATE RNA AND PROTEIN MOLECULES ARE LARGELY THE SAME IN PRINCIPLE BUT INVOLVE SLIGHTLY DIFFERENT TECHNIQUES BECAUSE OF THE UNIQUE PROPERTIES OF EACH CLASS OF MACROMOLECULE  IN 1975, E. M. SOUTHERN PUBLISHED AN IMPORTANT PROCEDURE THAT ALLOWED INVESTIGATORS TO IDENTIFY THE LOCATIONS OF GENES AND OTHER DNA SEQUENCES ON RESTRICTION FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS THE ESSENTIAL FEATURE OF THIS TECHNIQUE IS THE TRANSFER OF THE DNA MOLECULES THAT HAVE BEEN SEPARATED BY GEL ELECTROPHORESIS ONTO NITROCELLULOSE OR NYLON MEMBRANES  PROCEDURE USED TO TRANSFER DNAS SEPARATED BY GEL ELECTROPHORESIS TO NYLON MEMBRANES. THE TRANSFER SOLUTION CARRIES THE DNA FROM THE GEL TO THE MEMBRANE AS THE DRY PAPER TOWELS ON TOP DRAW THE SALT SOLUTION FROM THE RESERVOIR THROUGH THE GEL TO THE TOWELS. THE DNA BINDS TO THE MEMBRANE ON CONTACT. THE MEMBRANE WITH THE DNA BOUND TO IT IS DRIED AND BAKED UNDER VACUUM TO AFFIX THE DNA FIRMLY PRIOR TO HYBRIDIZATION. SSC IS A SOLUTION CONTAINING SODIUM CHLORIDE AND SODIUM CITRATE.  Southern blots SUCH TRANSFERS OF DNA TO MEMBRANES ARE CALLED SOUTHERN BLOTS AFTER THE SCIENTIST WHO DEVELOPED THE TECHNIQUE. THE DNA IS DENATURED EITHER PRIOR TO OR DURING TRANSFER BY PLACING THE GEL IN AN ALKALINE SOLUTION. AFTER TRANSFER, THE DNA IS IMMOBILIZED ON THE MEMBRANE BY DRYING OR UV IRRADIATION.  A RADIOACTIVE DNA PROBE CONTAINING THE SEQUENCE OF INTEREST IS THEN HYBRIDIZED WITH THE IMMOBILIZED DNA ON THE MEMBRANE. THE PROBE WILL HYBRIDIZE ONLY WITH DNA MOLECULES THAT CONTAIN A NUCLEOTIDE SEQUENCE COMPLEMENTARY TO THE SEQUENCE OF THE PROBE. NONHYBRIDIZED PROBE IS THEN WASHED OFF THE MEMBRANE, AND THE WASHED MEMBRANE IS EXPOSED TO X-RAY FILM TO DETECT THE PRESENCE OF THE RADIOACTIVITY. AFTER THE FILM IS DEVELOPED, THE DARK BANDS SHOW THE POSITIONS OF DNA SEQUENCES THAT HAVE HYBRIDIZED WITH THE PROBE  The ability to transfer DNA molecules that have been separated by gel electrophoresis to nylon membranes for hybridization studies and other types of analyses has proven to be extremely useful  Detection of a deletion of the X-linked androgen receptor gene by Southern blotting. The individual with androgen insensitivity has a 46,XY karyotype but is phenotypically female and therefore depicted by a circle in the pedigree. Left, all samples appear the same Right, After Southern blotting and hybridization to a cDNA probe for the human androgen receptor gene, the individual with androgen insensitivity syndrome is deleted for this gene (middle lane).  ANALYSIS OF RNAS BY NORTHERN BLOT HYBRIDIZATIONS IF DNA MOLECULES CAN BE TRANSFERRED FROM AGAROSE GELS TO NYLON MEMBRANES FOR HYBRIDIZATION STUDIES, WE MIGHT EXPECT THAT RNA MOLECULES SEPARATED BY AGAROSE GEL ELECTROPHORESIS COULD BE SIMILARLY TRANSFERRED AND ANALYZED. INDEED, SUCH RNA TRANSFERS ARE USED ROUTINELY IN GENETICS LABORATORIES.  NORTHERN BLOTS RNA BLOTS ARE CALLED NORTHERN BLOTS IN RECOGNITION OF THE FACT THAT THE PROCEDURE IS ANALOGOUS TO THE SOUTHERN BLOTTING TECHNIQUE, BUT WITH RNA MOLECULES BEING SEPARATED AND TRANSFERRED TO A MEMBRANE. THIS TERMINOLOGY HAS BEEN EXTENDED TO THE TRANSFER OF PROTEINS FROM GELS TO MEMBRANES, A PROCEDURE CALLED WESTERN BLOTTING.  THE NORTHERN BLOT PROCEDURE IS ESSENTIALLY IDENTICAL TO THAT USED FOR SOUTHERN BLOT TRANSFERS HOWEVER, RNA MOLECULES ARE VERY SENSITIVE TO DEGRADATION BY RNASES. CARE MUST BE TAKEN TO PREVENT CONTAMINATION OF MATERIALS WITH THESE EXTREMELY STABLE ENZYMES.  MOST RNA MOLECULES CONTAIN CONSIDERABLE SECONDARY STRUCTURE AND MUST THEREFORE BE KEPT DENATURED DURING ELECTROPHORESIS IN ORDER TO SEPARATE THEM ON THE BASIS OF SIZE. DENATURATION IS ACCOMPLISHED BY ADDING FORMALDEHYDE OR SOME OTHER CHEMICAL DENATURANT TO THE BUFFER USED FOR ELECTROPHORESIS. AFTER TRANSFER TO AN APPROPRIATE MEMBRANE, THE RNA BLOT IS HYBRIDIZED TO EITHER RNA OR DNA PROBES JUST AS WITH A SOUTHERN BLOT. ALLELE-SPECIFIC OLIGONUCLEOTIDE [ASO]  NORTHERN BLOT HYBRIDIZATIONS ARE EXTREMELY HELPFUL IN STUDIES OF GENE EXPRESSION. THEY CAN BE USED TO DETERMINE WHEN AND WHERE A PARTICULAR GENE IS EXPRESSED. NORTHERN BLOT HYBRIDIZATIONS ONLY MEASURE THE ACCUMULATION OF RNA TRANSCRIPTS. THEY PROVIDE NO INFORMATION ABOUT WHY THE OBSERVED ACCUMULATION HAS OCCURRED.  ANALYSIS OF PROTEINS BY WESTERN BLOT TECHNIQUES POLYACRYLAMIDE GEL ELECTROPHORESIS IS AN IMPORTANT TOOL FOR THE SEPARATION AND CHARACTERIZATION OF PROTEINS. BECAUSE MANY FUNCTIONAL PROTEINS ARE COMPOSED OF TWO OR MORE SUBUNITS, INDIVIDUAL POLYPEPTIDES ARE SEPARATED BY ELECTROPHORESIS IN THE PRESENCE OF THE DETERGENT SODIUM DODECYL SULFATE (SDS), WHICH DENATURES THE PROTEINS. AFTER ELECTROPHORESIS, THE PROTEINS ARE DETECTED BY STAINING WITH COOMASSIE BLUE OR SILVER STAIN. HOWEVER, THE SEPARATED POLYPEPTIDES ALSO CAN BE TRANSFERRED FROM THE GEL TO A NITROCELLULOSE MEMBRANE, AND INDIVIDUAL PROTEINS CAN BE DETECTED WITH ANTIBODIES.  WESTERN BLOTTING THIS TRANSFER OF PROTEINS FROM ACRYLAMIDE GELS TO NITROCELLULOSE MEMBRANES, CALLED WESTERN BLOTTING, IS PERFORMED BY USING AN ELECTRIC CURRENT TO MOVE THE PROTEINS FROM THE GEL TO THE SURFACE OF THE MEMBRANE.  AFTER TRANSFER, A SPECIFIC PROTEIN OF INTEREST IS IDENTIFIED BY PLACING THE MEMBRANE WITH THE IMMOBILIZED PROTEINS IN A SOLUTION CONTAINING AN ANTIBODY TO THE PROTEIN. NONBOUND ANTIBODIES ARE THEN WASHED OFF THE MEMBRANE, AND THE PRESENCE OF THE INITIAL (PRIMARY) ANTIBODY IS DETECTED BY PLACING THE MEMBRANE IN A SOLUTION CONTAINING A SECONDARY ANTIBODY.  THIS SECONDARY ANTIBODY REACTS WITH IMMUNOGLOBULINS (THE GROUP OF PROTEINS COMPRISING ALL ANTIBODIES) IN GENERAL. THE SECONDARY ANTIBODY IS CONJUGATED TO EITHER A RADIOACTIVE ISOTOPE (PERMITTING AUTORADIOGRAPHY) OR AN ENZYME THAT PRODUCES A VISIBLE PRODUCT WHEN THE PROPER SUBSTRATE IS ADDED.  KEY POINTS DNA RESTRICTION FRAGMENTS AND OTHER SMALL DNA MOLECULES CAN BE SEPARATED BY AGAROSE OR ACRYLAMIDE GEL ELECTROPHORESIS AND TRANSFERRED TO NYLON MEMBRANES TO PRODUCE DNA GEL BLOTS CALLED SOUTHERN BLOTS. THE DNAS ON SOUTHERN BLOTS CAN BE HYBRIDIZED TO LABELED DNA PROBES TO DETECT SEQUENCES OF INTEREST BY AUTORADIOGRAPHY.  KEY POINTS WHEN RNA MOLECULES ARE SEPARATED BY GEL ELECTROPHORESIS AND TRANSFERRED TO MEMBRANES FOR ANALYSIS, THE RESULTING RNA GEL BLOTS ARE CALLED NORTHERN BLOTS. RNA MOLECULES CAN BE DETECTED AND ANALYZED BY REVERSE TRANSCRIPTASE-PCR (RT-PCR). WHEN PROTEINS ARE TRANSFERRED FROM GELS TO MEMBRANES AND DETECTED WITH ANTIBODIES, THE PRODUCTS ARE CALLED WESTERN BLOTS. გმადლობთ , ყურადღებისთვის !!!