Phép lai “Southern” được đặt theo tên của Sir Edwin SouthernPhát triển vào năm 1975Một trong những ấn phẩm khoa học được trích dẫn nhiều nhấtGiúp Sir Southern đạt giải Lasker năm 2005 Đặt tên các phương pháp còn lại như là một kiểu chơi chữ theo Southern BlotSouthern blot : DNADNASử dụng gel điện di cùng với đầu dò lai đặc trưng cho mỗi đoạn cắt giới hạn của DNA genomic (hoặc DNA từ nguồn khác, chẳng hạn như plasmid)DNA xác định với trình tự base đặc trưngCó thể thực hiện để phát hiện những gene cụ thể tồn tại trong tế bào.Mục đích của phép lai SouthernCố định DNA vào một chất cố địnhMàng + chất nền giống giấy+ nylon hoặc nitrocellulose+ thường tích điện dương yếuNhận biết trình tự DNA (gene) quan tâmQuy trình chung của kỹ thuật Southern Blot
Heat shock Transformation Heat shock Transformation Electrophoration Transformation Southern blot • • ‘Southern’ hybridization named after Sir Edwin Southern Developed in 1975 • One of the most highly cited scientific publications • Earned Sir Southern a Lasker Award in 2005 Northern blot (RNA) Western blot (Protein) Eastern blot (???) Southern blot (DNA) Southern Blot: DNA-DNA* Uses gel electrophoresis together with hybridization probes to characterize restriction fragments of genomic DNA (or DNA from other sources, such as plasmids) Identifies DNA with a specific base sequence Can be done to detect specific genes present in cells Goals of Southern Hybridization • Immobilize DNA onto a permanent substrate • Membrane – paper-like matrix – nylon or nitrocellulose – usually has a slight positive charge • Identify DNA sequence (gene) of interest General Scheme for Southern Blot Southern Steps DNA to be analyzed is digested to completion with a restriction endonuclease Electrophoresis to maximally separate restriction fragments in the expected size range A set of standards of known size is run in one lane of the gel Blot fragments onto a nitrocellulose membrane Hybridize with the 32P probe Autoradiography 10 Average Restriction Fragment Length n = 4, 256 base pairs n = 6, 4096 base pairs n = 8, 65.5 kb base pairs How many genomic clones must be screened to find your gene? Theoretically, you will need to screen N clones where N=ln(1-P)/ln(1-f) where P=the probability of finding your gene and f=the average size of the cloned genomic sequence in your vector divided by the total genome size How many clones must you screen to find your gene in a human gene library packaged in EMBL with 99% certainty? N=ln(1-0.99)/ln(1-20kb/2.8 x 106kb)= 6.4 x 105 clones Genomic Sequences and Coverage N = ln(1 - P) ln(1 - f) N = number of clones P = probability of recovering a sequence, f = fraction of the genome of each clone E coli vs Humans # Clones = ln(1 - P) ln(1 - v/g) P = probability of including any one sequence v/g = insert size / genome size E coli Genome = 4.6 mb n = 4.6 mb / 20 kb insert = 230 P = 0.999 # Clones = 1585 Human Genome = 2900 mb n = 2900 mb / 20 kb insert = 145,000 P = 0.999 # Clones = 1,001,621 Generating A Genomic Library 1.Preparation of arms and genomic inserts Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes Infection and formation of plaques Screening procedures Screening procedures Screening Colony and plaque hybridization Expression screening Hybrid arrest and release Chromosome walking (repeat screening) Colony and plaque hybridization Transfer the DNA in the plaque or colony to a Nylon or nitrocellulose membrane Phage DNA bind to the membrane directly Bacterial colonies must be lysed to release DNA on the membrane surface Hybridization (in a solution (Alkali treatment) Containing Nucleic acid probe) X-ray film(radioactively labeled ) antibody or enzyme Wash to remove unhybri(modified nucleotide dization probe and visualize labeled Line up the hybridizated region or repeated hybridization Screening libraries Hybridization to identify the interested DNA or its RNA product Nucleic acid probe: In a short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA Probe labels • Radiolabels • Non-radioactive labels • Chemiluminescence: Fluorescence Chemicals: (FISH) • Antibodies Probe labels Radiolabels e.g 32 P, 35 S, 125 I, H Detection is by autoradiography Direct Non-radioactive labels FluorescentBiotin; “Reporter group “: Alkaline phosphatase and horseradish peroxidase Labeled probe Chemiluminescence Chemiluminescence: chemiluminescent chemicals attached to the probe are detected by Specimen DNA T F their light emission using a luminometer A A T Fluorescence Chemicals: attached to probe C T A G G A fluoresce under UV light-useful for the direct COVALEN G C T C examination of microbiological or cytological T BOND specimens under the microscope – a technique F known as fluorescent in situ hybridization (FISH) FISH Probe DNA Antibodies An antigenic group is coupled to the probe and its presence detected using specific antibodies Also, monoclonal antibodies have been developed that will recognize DNA-RNA hybrids Colony Hybridisation 88 Screening of Libraries Hybridisation: 90 Plaque Hybridisation 91 Screening of Expression Libraries with Antibodies Primary Antibody: against protein of interest (specific) Secondary Antibody: against proteins (antibodies) produced in rabbit, mouse, bird,… (unspecific but labeled) 92 ... Southern DNA on membrane Digest DNA Convert dsDNA to ssDNA Probe with DNA or RNA • • • • 23 Northern RNA on membrane No need to digest DNA Denature “folded” RNA with formaldehyde Probe with DNA or... blot (???) Southern blot (DNA) Southern Blot: DNA- DNA* Uses gel electrophoresis together with hybridization probes to characterize restriction fragments of genomic DNA (or DNA from other sources,... weight • ssDNA binds to nitrocellulose at same position it had on the gel • Vacum dry nitrocellulose at 80C to permanently fix DNA in place or cross link (via covalent bonds) the DNA to the