DNA Technology and Genomics Chapter 20 DNA Technology and Genomics

DNA Cloning

Restriction enzymes Where are restriction enzymes found? In bacterial cells, to help fight viruses What is the difference between blunt and sticky ends? What is a restriction site? The area that the restriction enzyme recognizes Why are restriction sites often palindromes? A MAN A PLAN A CANAL PANAMA

Cloning The original plasmid is called the cloning vector After insertion of the gene of interest into the plasmid the plasmid is called Recombinant DNA How would the recombinant plasmid that we engineered in class be used in the lab?

Identifying the gene of interest Use different probes How would you identify different genes?

DNA Libraries Shotgun approach makes thousands of different recombinant plasmids This set of plasmid clones is called a Besides using plasmids scientists use as cloning vectors genomic library bacteriophages

cDNA Library

Different libraries have different uses Which library would you use in the following scenarios? You want to clone a gene, without knowing where and how it is expressed. You want to know regulatory sequences. You want to know the coding sequences of a gene. genomic library genomic library cDNA library

Expressing Eukaryotic Genes How can scientists get bacterial cells to express eukaryotic genes? Insert the desired gene with a prokaryotic promoter attached. How do they remove introns? Insert cDNA in the first place. Use YACs (Yeast artificial chromosomes)…eukaryotic hosts

DNA Amplification PCR = polymerase chain reaction PCR Animation

Gel electrophoresis Uses to separate DNA DNA has a charge. DNA moves towards the electrode. DNA molecules of sizes move the furthest through the gel. electrical current negative positive smaller

RFLP Restriction enzymes DNA. Fragments that result are of sizes. These differently sized fragments are called . RFLP’s are found on noncoding segments of DNA and are due to . cut different RFLP’s VNTR’s

Southern Blotting Technique

Uses recombinants Mapping the Genome Uses restriction fragments

Sanger Sequencing

Shotgun Approach

Identifying Protein Coding Genes What would you look for if you wanted to find a protein coding gene? Start and stop codons These sequences are called EST’s (expressed sequence tags)

Genome Sizes

Genomes If there is no pattern between complexity and number of genes what makes organisms more complex? More involved regulation Alternative splicing

Determining Gene Function How can we determine the function of different genes? RNAi; silence a gene and see what it did Insert synthetic double stranded RNA’s that will activate dicer. This was used to identify the function of C. elegans genes

Genome wide expression Resulting colors shows which genes were being expressed in each tissue Use two tissue samples with differently colored fluorescent nucleotides

Comparing Genomes Has revealed many similar regions even between yeast and us. Can be used to derive evolutionary relatedness. Can use differences between closely related species to explain phenotypic differences. (chimps and humans with regard to language acquisition). Has led to proteomics.

Practical Applications Medical Disease diagnosis Gene Therapy Medicines Forensics Environmental Cleanup Aggriculture Pharm animals GMO foods/plants

Safety and Ethics What are the fears? Is this ethical? Is it too fast? Is it really that good in the long run?