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Inject es cells into early embryos,. Transfer embryos to foster mother,. Breed chimeric mice and screen for progeny with mutant germ line,. Screen progeny dna for mutation,. Mate diary heterozygotes (X/x-. Screen progeny dna for ko genotype (X-/X-). Entire process takes a year. Microarrays detect expressed genes by hybridization Each spot has a different synthetic oligonucleotide complementary to a specific gene. Label cDNAs with red fluorophore in one condition and green fluorophore in another reference condition.
Transform -ch3 -ch3 CH3- pcr -ch3 -ch3 CH3- -ch3 Dpn1 Digested Gene replacement in mice - make donor cells. Insert drug markers into genome of es cells. Select to enrich for homologous recombinants Check insertion site by southern blotting supermarket neor confers resistance to g-418. Tkhsv confers sensitivity to ganciclovir. Southern blotting detects dna sequences by hybridization Digest dna using restriction enzyme(s) Run gel Transfer dna from gel to (nitrocellulose) paper. Denature dna, hybridize probe dna, and wash off excess probe. Detect the probe on the paper. Northern blotting detects rna on the gel. Gene replacement in mice - germline incorporation Transgenic mice express a new gene Which mouse expresses extra copies of the growth hormone gene?
Create clusters: Attach one end to a solid support, pcr in situ with one primer attached to support pcr deblock 3 oh and remove color. Repeat synthesis of next base. Repeat 30 x for 106 clusters! Assemble genome sequence by finding overlaps of 30mers and comparing to known genome sequence. Ssdna cluster Genome resequencing Currently: 109 reads 5,000 x-prize: Human genome 1,000 (3.2 x 109 bases (x 8 reads) Site-directed mutagenesis CH3- -ch3 Denature methylated template and anneal divergent mutagenic primers. Pcr amplify the entire plasmid with a dna pol lacking 5- 3 exonuclease. Select against parental strands with Dpn1 restriction enzyme, which cuts methylated dna.
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Dna methods summary restriction enzymes cut at specific dna sites. (N) pcr amplifies any target dna sequence. (N) Genes and genomes can be sequenced by chain termination. (N) Oligonucleotides can be used to change bases by site-directed mutagenesis. (N) southern blotting detects sequences by hybridization.
(N) Microarrays detect gene expression patterns over the genome. Dna sequencing by partial chain termination All fragments start at the primer All fragments ending in a particular base have a different length and a different color tag Separating the mixture of products by size reveals the sequence. 1000 bases/reaction Genome resequencing - many short reads. Prepare sample: Shear, repair, ligate adapters to both ends. Sequence-by-synthesis: Denature, add primer all 4 fluorescent dNTPs with blocked 3 oh to add 1 base to each peer cluster. Random dna fragment read each cluster with laser.
The ends should match for efficient ligation. Cloning without dna ligase, gateway cloning, ligation-independent cloning. Prepare open vector and insert with the same long sticky ends pol i klenow fragment datp mix and let the ends anneal. Transform the nicked plasmid. The plasmid is repaired in vivo. Prepare an insert flanked by sites for a site-specific dna recombinase.
Mix insert with the closed vector containing the recipient recombination site and recombinase enzyme. No dT in template insert 20 base pairs ector Insert vector Insert Vector Vectors allow dna sequences to be cloned -3 Transient transfection: eukaryotes Stable transfection Plasmid is unstable - expression variable Plasmid integrated in large tandem arrays - protein overexressed Chemical dna synthesis Sequential. 3 5 Frontiers in dna synthesis Currently: 100-200 nucleotides routine (Assemble 5 kB) 10,000 largest. Primer set for the human genome (30,000 genes) 104 goal 1: make yeast chromosome 3: 300 kb without errors! (Jeff boeke; 300,000) goal 2: Assemble a total of 16 X 106 w/o errors for 1000 (George Church) pcr (Polymerase Chain reaction isolate and amplify any dna sequence copies: 1 2 4 8 N cycles amplifies the target sequence 2N-fold. Quantitative pcr (qpcr) defines amount of starting template.
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Genes can be knocked out(deleted) or replaced in prokaryotes and eukaryotes. Microarrays detect gene expression patterns over the genome. Restriction enzymes cut dna at specific sites 3 types of ends: barbing 5 overhang, blunt and 3 overhang. Cognate methyl transferases protect host genome from digestion. Restriction-modification systems degrade foreign dna. Average frequency of restriction sites in random dna sequences, average frequency 1/256 1/4,096 1/65,536, site size 4 6 8 (1/4 x 1/4 x 1/4 x 1/4). The average occurrence of each sequence 1/4n, where n the site length and all bases are equally represented. A simple cloning procedure, cut insert and vector dna with a restriction enzyme. Mix and join ends with dna ligase.
While downloading, if for some reason you are not able to download a presentation, the homework publisher may have deleted the file from their server. Presentation Transcript, dNA methods summary, restriction enzymes cut at specific dna sites. Vectors allow genes to be cloned and proteins expressed. Gel electrophoresis separates dna on the basis of size. Pcr amplifies any target dna sequence. Genes and genomes can be sequenced by chain termination. Oligonucleotides can be used to change bases by site-directed mutagenesis. Southern blotting detects sequences by hybridization.
basis of size. Dnas can be synthesized (up to 100 bases commercially). Copyright Complaint Adult Content Flag as Inappropriate. I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described. Download Presentation, an Image/Link below is provided (as is) to download presentation. Download Policy: Content on the website is provided to you as is for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.
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