DNA-based information technologies Understand basics steps/enzymes/features of DNA cloning

1. DNA-based information technologies Understand basics steps/enzymes/features of DNA cloning Know main types of cloning vectors used - pros & cons of each Understand use of “probes” to identify DNA sequences Understand use of expression plasmids to study gene products Understand how site-directed mutagenesis is done and why it is useful to study proteins Know types of DNA libraries created & used

2. DNA cloning - separating a specific gene or DNA segment from a larger chromosome, attaching it to a small molecule of carrier DNA and then replication 5 general procedures of genetic engineering/recombinant DNA technology: 1. Cutting DNA at specific locations - restriction endonucleases 2. Selecting a small molecule of DNA capable of self-replication - cloning vectors 3. Joining two DNA fragments covalently - recombinant DNAs 4. Moving recombinant DNA from test tube to a host cell 5. Selecting or identifying host cells that contain recombinant DNA

4. RESTRICTION ENDONUCLEASES - protein enzymes that cleave the phosphodiester bonds that connect the nucleotide units in DNA or RNA at very SPECIFIC sites These enzymes are mainly produced by bacteria where they degrade invading foreign DNA (bacterial DNA protected by methylation of its DNA); REs have been purified from these sources and are now available commercially Most restriction enzymes recognize a specific sequence of 4-6 nucleotides in DNA and each will cut the DNA into discrete pieces known as restriction fragments

6. May need to purify DNA by gel electrophoresis/HPLC

12. Cloning vectors - plasmids, bacteriophage, BACs/YACs (HACs) Plasmids Circular DNA molecules that replicate separately from the host Introduced into bacterial cells by transformation or electroporation Naturally occurring plasmids ~5000-400,000 bp Features of a plasmid: 1. Origin of replication 2. Antibiotic resistance for selection 3. Unique restriction sites 4. Small size facilitates entry into cell Difficult to transform large plasmids into bacterial cells Difficult to clone DNA segments >15,000 bp when plasmids are used as the vector

18. Cloning vectors - plasmids, bacteriophage, BACs/YACs Bacteriophage Infects bacteria Can accommodate larger segments of DNA than plasmids Features of bacteriophage: 1. ~1/3 of its genome (48,502 bp) is nonessential and can be replaced with foreign DNA 2. DNA is packaged into infectious phage particles only if it is between 40,000 and 53,000 bp long - this ensures the packaging of recombinant DNA only

19. Infect bacteria

20. Cloning vectors - plasmids, bacteriophage, BACs/YACs Bacterial Artificial Chromosomes (BACs) Plasmids designed for the cloning for very long DNA segments (100,000 - 300,000 bp) Features of BACs: 1. Must have selectabel marker 2. Must have very stable origin of replication Use electroporation to get BACs into cells

21. Par genes - Assist in even distribution of plasmids to daughter cells at cell division Low copy number plasmid - this limits the opportunities for unwanted recombination Lac Z gene - b-galactosidase Blue-white colony screening Substrate - X-gal

22. Cloning vectors - plasmids, bacteriophage, BACs/YACs Yeast Artificial Chromosomes (YACs) Eukaryotic organism for the cloning for long DNA segments Most used - S. cerevisiae (14 x 106 bp) - sequence known Easy to grow and maintain Features of YACs that allow them to be maintained as a eukaryotic chromosome in the nucleus: 1. Yeast origin of replication 2. Two selectable markers 3. Specialized sequences (telomere & centromere) needed for stability and proper chromosomal segregation

23. Cloning vectors - YACs Telomeres - sequences at the ends of chromosomes that help stabilize Yeast have 100 bp of imprecisely repeated sequences: (5’)-(TxGy)n; x~1, y~4, n~20-100 Sequence lost here each round of replication - telomerase Centromeres - DNA sequence that functions during cell division as an attachment point for proteins that link the chromosome to the mitotic spindle Essential for the equal and orderly distribution of chromosome sets to daughter cells

24. Genomic fragments are separated by pulsed field gel electrophoresis DNA fragments can be up to 2 x 106 bp Stability of YAC clones increases with with size up to a point Inserts >150,000 bp stable Inserts <100,000 bp are gradually lost

27. Cloning - specific DNA detection by hybridization Sequence-based process for detecting a particular gene - use of probes

28. Cloning - specific DNA detection by hybridization Design of probe??

29. Cloning - protein expression Presence of correct sequence environment for eukaryotic DNA - expression vectors

30. Cloning - study of function of proteins using site-directed mutagenesis

32. From genes to genomes - creation of DNA libraries DNA library - collection of DNA clones gathered together as a source of DNA for sequencing, gene discovery, or gene function studies Genomic library - produced when complete genome of a particular organism is cleaved into thousands of fragments and all fragments are cloned by insertion into a cloning vector Using probes, order clones in a library to identify overlapping sequences Set of overlapping clones represents a long continuous segment of genome called a CONTIG Known sequences in a library are called sequence-tagged sites (STS) and aid in genomic sequencing projects

33. From genes to genomes - creation of DNA libraries Currently used libraries include genes that are expressed in an organism Create cDNA from transcribed RNAs - clone into vector - creation of cDNA library Aid for mapping of large genomes - cDNAs in a library are partially sequenced to produce a useful STS called an Expressed sequence tag (EST)

34. From genes to genomes - creation of DNA libraries cDNA library made more specialized by fusing a reporter gene to cDNA sequence GFP (green fluorescent protein)fused to allow study of location and movement of protein

35. From genes to genomes - creation of DNA libraries Use of PCR to amplify specific DNA sequences