3REKOMBİNASYONRekombinasyon: Yenibileşim - yenidenoluşum. Bir molekülün-hücrenin, atasal “wild type” yada ilkin(orijinal) yapısından farklılık göstermesi durumudur.I - In vivo rekombinasyonII- In vitro rekombinasyon
4Klon (Clone); Bir tek atasal diploid hücreden mitoz bölünme yoluyla birden fazla hücre eldesine denir. Rekombinant DNA teknolojisi ile sentezlenen identik DNA/gen kopyalarına denir. ”Gene cloning”
5Genetik Klonlamada Tarihçe Gelişme Araştırıcı Yıl Deniz hayvanlarında döllenme O.Hertwig İlk kez anne rahmi dışında döllenme L.Schenk Tüp ortamında insan yumurta hücresi döllendi MF Menkin Dondurulmuş sperm ile inek yumurtası döllendi Deney tüpünde döllenen bir memeli yavru doğdu Dondurulmuş embriyodan yavru fareler elde edildi Louise Brown isimli bebek deney tüpünde döllendi anne rahmine yerleştirilerek sağlıklı doğum yaptırıldı Avusturalyada donmuş embriyodan sağlıklı bir kız çocuğu elde edildi
6Genetik Klonlamada Tarihçe Gelişme Yıl Kiralık anne Mary Beth bebeğini vermeyi redetti Embriyo hücrelerinin çoğaltılmasıyla çok sayıda kuzu elde edildi İnsan embriyosu klonlandı çok tepki aldı J.Hall Dolly klonlandı I.Wilmut Fransada bir dananın 63 klonu elde edildi Totipotent stem hücrelerinden deneysel organogenezis Farede gen klonlama yöntemiyle insan kulağı gelişimi sağlandı 2001 Avusturalyada bir at klonlama ile Coada eşek doğurdu Amerikada ex vivo yapay rahim geliştirildi
7Klonlama Tipleri DNA /Gen düzeyinde klonlama Hücre düzeyinde klonlama Organizma /çekirdek düzeyinde klonlama
8Başarılı Klonlama Yapabilmek İçin Gen; Bağımsız olarak replike olabilmeli Konak hücreye kolaylıkla transfer edilebilmeli Seleksiyona olanak tanımalı
9Memeli Hücrelerine Gen Transfer Teknikleri Microinjection*** DAAE-Dextran Mediated Electroporation Lipofection Calcium Phosphate*** Protoplast Fusion Polyprene Viral infection*** (Lentivirus, Retrovirus, Adenovirus)*: Yaygın kullanılan yöntemler
18Nükleer Transplantasyon Wilmut ve arkadaşları donör hücre olarak 6 yaşında sağlıklı bir koyunun meme epitel hücresi ve resipient hücre olarak ise aynı koyunun metafaz II evresinde bekletilmiş enucleated yumurta hücresi kullandılar. Klonlama sonrası elde edilen ve annesiyle %100 aynı genotip ve fenotipte olan sağlıklı kuzuya DOLLY adını verdiler.
21Nükleer Klonlamanın Önemi Yumurta hücresinin embriyogeneziste spermden farklı artı(+) öneminin olduğu, Ökaryotik hücrenin G0 evresinde totipotent kromatin organizasyonu kazandıği, Metafaz II evresinde yumurta hücresinin klonlama için en uygun stage olduğu, Memelilerde eşeysiz üremenin mümkün olduğu, Bir gen yerine çekirdeğin tamamının transplante olabileceği gösterildi
22Klonlama Sonrasında; Unipotent hücrenin totipotent hücreye dönüştürülmesi, Sinir hücrelerinin rejenerasyonu, Telomerlerde ” end replicatin problem”giderilerek, yaşlanmanın geciktirilmesi, Stem hücrelerinden spesifik doku eldesi, Epigenetik modifikasyonu ile kanser tedavisine yeni bir yaklaşım,“Ex vivo gene replacement” ile genetik tedavi ve İnsan genom projesi önemli bir ivme kazanmıştır.
24Goals of DNA Technology Isolation of a particular gene or sequenceProduction of large quantities of a gene productProtein or RNAIncreased production efficiency for commercially made enzymes and drugsModification/improvement of existing organismsCorrection of genetic defects
25Amplifying DNAOften we need large quantities of a particular DNA molecule or fragment for analysis. Two ways to do this:-1. Insert DNA mol. in a plasmid and let it replicate in host >>> many identical copies (= ‘DNA cloning’)2. Use PCR technique - automated multiple rounds of replication >>> many identical copies.
26DNA CloningPurpose:- to amplify (bulk up) a small amount of DNA by inserting it into in a fast growing cell e.g. bacterium, so as bacterium divides we will have many copies of our DNA1. Obtain a DNA vector which can replicate inside a bacterial cell (plasmid or virus) which2. Insert DNA into vector - use restriction enzyme3. Transform host cells i.e. insert vector into host cell (e.g. bacterium)4. Clone host cells (along with desired DNA)5. Identify clones carrying DNA of interest
27The DNA of interest must be inserted into the vector. Vectors are convenient carriers of DNA. They are often viruses or plasmids.Usually are small circular DNA molecules and must be capable of replicating in the host cellA famous plasmid. The circular moleculein this electron micrograph is pSC101,the first plasmid used successfully to clonea vertebrate gene. Its name comes fromthe fact that it was theone-hundred-and-first plasmid isolated byStanley Cohen.The DNA of interest must be inserted into the vector.
28Restriction Enzymes Target or recognition sequence Cuts hereRestriction enzymes (R.E.) recognise target sequences and cut DNA in a specific manner.This R.E. leaves TTAA single stranded ends (‘sticky ends’)If you cut DNA of interest and plasmid with same restriction enzyme then you will have fragments with identical sticky ends.
29Sticky ends will readily rejoin - so its possible to join 2 DNA’s from different sources AATTTTAAPlasmids are usually chosen to have only one target site. DNA of interest can then insert into this siteRecombinant plasmid
30Transformation of host and selection of desired clones Bacteria are made to take up the recombinant plasmid & grown (cloned) in large numbers (TRANSFORMATION)Bacteria carrying desired sequence can be selected.Large amounts of DNA or proteins can be extracted
32Making a Genomic Library Genomic library = a complete collection of DNA fragments representing an organism’s entire genome.1. Cut up genome into thousands of fragments with an R.E.3. Result - a collection of bacterial colonies (clones) carrying all the foreign DNA fragments i.e. a genomic library2. Insert each of these into separate plasmids and then into separate host cells.
33A question for you - how will a cDNA library differ from a genomic library ? Which would have more genes ?What would be present in the clones in each case?Promoters ?EnhancersIntrons ?Poly-T (from poly-A tail)?
34How do we identify DNA mols. of different sizes ? long DNAshort DNAGel ElectrophoresisStandards of known M.W.Run DNA fragments through a gel under influence of an electric current. Each of the DNA fragments travels through the gel at a constant speed appropriate for its size.Longer molecules move more slowly so don’t travel as far.See Fig 20.8
35Polymerase Chain Reaction (PCR) Small amount of DNA can be amplified greatly - automated process involves:-A DNA polymerase which is stable at high temperaturesspecific primers to start off replication at known position.Three step cycle:Heat to separate DNA strands = DenaturationCool and allow primers to bind (Annealing)Polymerize new DNA strands (Extension)Repeat steps 25 – 35 times >>> millions of copies of original DNA
45Bacterial PlasmidsPlasmids are small, circular DNA molecules in bacteria.By inserting genes into plasmids, scientists can combine eukaryotic and prokaryotic DNA. (Recombinant DNA)Bacterial cells continually replicate the foreign gene along with their DNA.Cloning using plasmids can be used to:Identify a particular protein a gene makes (ie: for study)Produce large amounts of a particular protein/gene (ie: for use in medicine)
46Restriction Enzymes Also used to make recombinant DNA. Specifically cut DNA molecules at precise base locations.(restriction)
50Why Use Bacteria as vectors? Plasmids are easy to use to manipulate which genes are expressed in clones.2. Bacteria replicate very quickly and allow you to produce a large number of a desired gene.
51Identifying ClonesNot all of the reproduced bacteria are clones carrying the desired gene.Two ways to identify which are clones:Look for the geneLook for the protein the gene codes for
52Nucleic Acid Hybridization If you know the sequence of the cloned gene you are looking for, you can make a nucleic acid probe with a complementary sequence.The probe is radioactively labeled and allowed to base pair with the denatured (separated strands) DNA.The probes H-bond with their complement (cloned gene), thus identifying the cloned cells.Identified cells are cultured to produce more.
53Figure 20.4 Using a nucleic acid probe to identify a cloned gene
54Expressing Euk. Proteins in Bacteria It is more difficult to get the bacteria to translate the proteins because of differences in promotor sequences b/t prokaryotes and eukaryotes.Expression vectors are plasmids that contain the promotor sequence just before the restriction site.This allows the insertion of a eukaryotic gene right next to the prokaryotic promotor.
55Expressing Euk. Proteins in Bacteria Bacteria also lack the enzymes needed to remove introns from DNA.Therefore, cDNA (no introns) is inserted into plasmids to allow expression of the eukaryotic gene.Reverse transcriptase is the enzyme used to make cDNA from a fully processed mRNA strand.
56Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene
57Another Solution: Use Yeast (eukaryotic) Why?They grow quickly like bacteriaThey are eukaryotes (similar enzymes, metabolic mechanisms, protein mods)They have plasmids (rare for eukaryotes)Can replicate artificial chromosomes as well as DNA in plasmids
58Genomic LibrariesPlasmids and phages used to store copies of specific genes.
60PCR Faster and more specific method for amplifying short DNA sequences After DNA is denatured (split), primers start new complementary strands with each strand producing more molecules of the sequence.In vitro = doesn’t require living cellsIn test tube: denatured DNA, free nucleotides, DNA primers (specific to gene desired), “special” DNA polymerase (can withstand high heat w/o denaturing)
61Analyzing DNAGel electrophoresis separates molecules based on size, charge, density, etc.Linear DNA – mainly separated by fragment length (size)Molecules of DNA are separated into bands of molecules of the same length.
65Southern BlottingProduce restriction fragments of DNA (restriction enzyme used)Separate fragments (gel electrophoresis)BlottingTransfer DNA to nitrocellulose paperHybridize with radioactive probesAutoradiography to identify which have probes.
66RFLPsPolymorphisms that result from differences in noncoding regions of DNA.Restriction enzymes cut DNA into different fragments in each variant.RFLP markers allowed scientists to more accurately map the human genome.Genetic studies do not have to rely on phenotypic (appearance/proteins) differences to guide them anymore.
67In Situ (on a slide) Hybridization Radioactively (or fluorescently) labeled probes base pair with complementary denatured DNA on a microscope slide.Autoradiography and staining identify the location of the bound probe.
68Human Genome ProjectAttempt to map the genes on every human chromosome as well as noncoding information.Three stagesGenetic Mapping (linkage)Physical MappingGene (DNA) SequencingGenomes of species that give insight to human codes are also being done (fruit fly, E coli, yeast)
69Genetic MappingLinkage maps based on recombination frequencies created.Linkage maps portray gene sequences as you physically move along a chromosome.Genetic markers along the chromosome allow researchers to use them as reference points while studying other genes.
70Physical MappingDetermines the actual distance between the markers along a chromosome (# of bases)Utilizes chromosome walking to identify the distance between.Use a series of probes to identify the DNA sequence of various restriction fragments, and ultimately the entire length of DNA sample.
72DNA SequencingAs of 1998, 3% of the human genome had been sequenced using automation. (Sanger Method)Once the sequences of all the genes are known, scientists can begin to study all of their functions, and manipulate their products in many ways.
73Applied Genetics Diagnosis of Genetic Disorders Sequence individuals before birth to know if their DNA contains abnormalitiesHuman Gene TherapyReplace missing or fix damaged genes in affected individuals
75Pharmaceuticals Hormone production (ie: Human Growth) Protein supplementsHIV treatment: “decoy” receptor protein used to inhibit HIV virus’ ability to enter cellVaccinesProteins that stimulate immune response can be used instead of traditional vaccinesAntisense Nucleic AcidsBlock translation of certain proteins
76Other Uses of DNA Tech DNA Fingerprinting for forensic cases Environmental cleanupAgricultureAnimal HusbandryGenetic Engineering of Plants
77The Future of GeneticsThe future of science lies in genetics???
79MicroarraysSee FigAll known genes are spotted on a small solid support (chip). Many uses e.g.A specific cDNA is tagged with a fluorescent marker and hybridized to the array
80MicroarraysThe cDNAs would bind only to those genomic clones that have complementary DNA sequencesThese clones would “light up”Have been used for example to look at cancer cells - which genes are turned ON or OFF compared to normal cells ?
81DNA SequencingUses dideoxy nucleotides to terminate replication of a chain at a known base.Automatic sequencing of DNA
82Chain termination by dideoxynucleotides Normal nucleotidesNew nucleotide can be added on to this 3’ endDideoxy nucleotideNew nucleotide can NOT be added on to this 3’ end
83Dideoxy sequencing All essential components of DNA synthesis are required, namely...DNA polymerase…plus ddNTPs5
85Dideoxy sequencingResult - a series of DNA fragments each 1 base longer than the next and terminating in a specific ddbase e.g ddT or ddGThis ddbase is the complementary one to the one on the strand being sequenced e.g ddT would be opposite A
86Dideoxy sequencing Heat the mixture to separate the dd-terminated strands from the templates
87Dideoxy sequencingddRibo-terminated, fluorescent DNAs are separated by size using gel electrophoresisBases color coded - easy to read sequence.Sequence here is (from bottom) CCTAGGAATCC-+1
88Vast amounts of data sequences now on computer accessible by online data banks. Already many complete genomes sequenced.DNA Sequencer machines read the fluorescence of each band - store the sequence in computers
89Genome Sizes and Numbers of Genes OrganismGenome SizeEstimated Number of GenesH. influenzae(bacterium)1.8 Mb1,700S. cerevisiae(yeast)12 Mb6,000C. elegans (nematode)97 Mb19,000A. thaliana (plant)100 Mb25,000D. melanogaster (fruit fly)180 Mb13,000H. sapiens (human)3,200 Mb30,000 – 40,000
90Southern BlottingUsed to check for the presence of a specific DNA sequence in a mixture of DNA fragments.1. Separate the mix of DNA fragments by electrophoresis2. Add a labeled DNA probe. It will attach to a complementary sequence (if present)DNA probe3. The label will make this band ‘light up’
91Northern and Western blotting Southern blots identify a specific DNA sequence in a mix of DNAsIn a similar way:-Northern blots identify a specific RNA sequence in a mix of RNAsWestern blots identify a specific protein sequence in a mix of proteins
92In-situ hybridization In situ hybridization - probes can bind to specific sequences on a chromosome in a cell prep. - show where it is locatedThis involvesthe use of DNA or oligonucleotides complementary to a nucleotidesequence on the chromosomes. These "cDNA probes" areconjugated to fluorescein, rhodamine, Texas red, or anotherfluorescent compound and then used to detect the sites of the geneon the individual chromosomes. This figure detects a sequence onchromosome 21 in a normal individual (white label). Note, there aretwo copies of this chromosome, each bearing the sequence. Thisprobe is useful for the detection of Down syndrome which containsan extra chromosome 21.