DNA Cloning Goal is to generate large amounts of pure DNA that can be manipulated and studied.

1. Chapters 8, 9, 10 - Recombinant DNA Technology and Other Molecular Methods Used in Genomic Analysis • Lots of topics to be covered including: • Recombinant DNA • Restriction enzymes – utility & analysis • Library construction • PCR – conventional & real-time • DNA sequencing – dideoxy & next generation • Genomic analysis • Today I want to discuss 3 important topics: • The process of cloning DNA in E. coli • Utility of restriction enzymes • Recombinant DNA libraries – genomic & cDNA

2. Cloning technology - generation of many copies of DNA template (e.g., recombinant DNA molecule) that is replicated in a host. • Developed in the 1970s. • Can generate un unlimited supply of gene copies. • Many applications: • Genetic mapping • DNA sequencing • Mutation studies • Transformation • Genetic engineering • Genome sequencing • Prior to the development of this technology, DNA was difficult to work with because it was hard to obtain sufficient copy number to visualize or manipulate.

3. DNA Cloning • Goal is to generate large amounts of pure DNA that can be manipulated and studied. • DNA is cloned by the following steps: • Isolate DNA from organism (e.g., extract DNA) • Cut DNA with restriction enzymes to a desired size. • Splice (or ligate) each piece of DNA into a cloning vector to create a recombinant DNA molecule. Cloning vector = artificial DNA molecule capable of replicating in a host organism (e.g., bacteria). • Transform recombinant DNA (cloning vector + DNA fragment) into a host that will replicate and make copies. • E. coli is the most common host.

4. Step 1-Isolate whole genomic DNA from organism DNA extraction easily performed using: • SDS (detergent) to break up cell membrane and organelles. • Salt (NaCl) lyses cells and binds the DNA strands together. • Proteinase K to digest proteins bound to DNA (essential to remove eukaryotic chromatin). • Ethanol (EtOH) to precipitate and wash DNA. • Water to resuspend and store DNA. Storage - DNA can be stored short-term at room temperature, but is best stored long-term at -80C or in liquid nitrogen. Ideal storage conditions - preserve large uncut DNA fragments. Suboptimal storage conditions - preserve smaller DNA fragments. *Average size of DNA fragments is important for applications involving large regions of DNA sequence/less important for applications involving short regions of DNA sequence.

5. Step 2-Cut DNA with restriction enzymes Restriction enzymes recognize specific bases pair sequences in DNA called restriction sites and cleave the DNA by hydrolyzing the phosphodiester bond. • Cut occurs between the 3’ carbon of the first nucleotide and the phosphate of the next nucleotide. • Restriction fragment ends have 5’ phosphates & 3’ hydroxyls. restriction enzyme

6. Step 2-Cut DNA with restriction enzymes (cont.) • Most restriction enzymes occur naturally in bacteria. • Protect bacteria against viruses (bacteriophages) by cutting up viral DNA. • Bacteria protects their DNA by modifying possible restriction sites (methylation). • More than 400 restriction enzymes have been isolated. • Names typically begin with 3 italicized letters. EnzymeSource EcoRI E. coli RY13 HindIII Haemophilus influenzae Rd BamHI Bacillus amyloliquefaciens H • Many restriction sites are palindromes of 4-, 6-, or 8-base pairs. • Short restriction site sequences occur more frequently in the genome than longer restriction site sequences. • Their frequency is a function of (1/4)n

7. Fig. 8.1, EcoRI “6-base cutter”

8. Step 2-Cut DNA with restriction enzymes (cont.) • Some restriction enzymes produce blunt ends, whereas others produce sticky (overhanging staggered) ends. • Sticky ends are useful for DNA cloning because complementary sequences anneal and can be joined directly by DNA ligase without using ‘adapters’. Fig. 8.2

9. Fig. 8.3, Cut and ligate 2 DNAs with EcoRI ---> recombinant DNA

10. Step 3-Splice (or ligate) DNA into some kind of cloning vector to create a recombinant DNA molecule Six different types of cloning vectors: • Plasmid cloning vector • Phage  cloning vector • Cosmid cloning vector • Shuttle vectors • Yeast artificial chromosome (YAC) • Bacterial artificial chromosome (BAC) • Fosmid cloning vector

11. Plasmid Cloning Vectors: • Bacterial plasmids, naturally occurring small ‘satellite’ chromosome, circular double-stranded extrachromosomal DNA elements capable of replicating autonomously. • Plasmid vectors engineered from bacterial plasmids for use in cloning. • Feeatures (e.g., E. coli plasmid vectors): • Origin sequence (ori) required for replication. • Selectable trait that enables E. coli that carry the plasmid to be separated from E. coli that do not (e.g., antibiotic resistance, grow cells on antibiotic; only those cells with the anti-biotic resistance grow in colony). • Unique restriction site such that an enzyme cuts the plasmid DNA in only one place. A fragment of DNA cut with the same enzyme can then be inserted into the plasmid restriction site. • Simple marker that allows you to distinguish plasmids that contain inserts from those that do not (e.g., lacZ+ gene)

12. Fig. 8.4, pUC19 Polylinker: restriction sites lacZ+ gene Origin sequence Ampicillin resistance gene

13. Detailed map showing polylinker region in pUC57 (genecript.com)

14. Fig. 8.5 *Cut with same restriction enzyme *DNA ligase

15. Some features of pUC19 plasmid vector: High copy number in E. coli, ~100 copies/cell, provides high yield. Selectable marker is ampR. Ampicillin in growth medium prevents growth of all other E. coli that do not contain plasmid. Cluster of several different restriction sites called a polylinker occurs in the lacZ (-galactosidase) gene. Cloned DNA disrupts reading frame and -galactosidase production. Add X-gal to medium; turns blue in presence of -galactosidase. Plaque growth: blue = no inserted DNA and white = inserted DNA. Some % of digested vectors will reanneal with no insert. Remove 5’ phosphates with alkaline phosphatase to prevent recircularization (this also eliminates some blue plaques). Plasmids are transformed into E. coli by chemical incubation or electroporation (electrical shock disrupts the cell membrane). Good for <10kb; Cloned inserts >10 kb typically are unstable.

16. Electroporation:

18. Phage  cloning vectors: Engineered version of bacteriophage  (infects E. coli). Central region of the  chromosome (linear) is cut with a restriction enzyme and digested DNA is inserted. DNA is packaged in phage heads to form virus particles. Phages with both ends of the  chormosome and a 37-52 kbinsert replicate by infecting E. coli. Phages replicate using E. coli and the lytic cycle (see Fig. 3.13). Produces large quantities of 37-52 kb cloned DNA. Like plasmid vectors, large number of restriction sites available; phage  cloning vectorsuseful for larger DNA fragments than pUC19 plasmid vectors.

20. Cosmid cloning vectors: Features of both plasmid and phage cloning vectors. Do not occur naturally; circular. Origin (ori) sequence for E. coli. Selectable marker, e.g. ampR. Restriction sites. Phage  cos site permits packaging into  phages and introduction to E. coli cells. Useful for 37-52 kb.

21. Shuttle vectors: Capable of replicating in two or more types of hosts.. Replicate autonomously, or integrate into the host genome and replicate when the host replicates. Commonly used for transporting genes from one organism to another (i.e., transforming animal and plant cells). Example: *Insert firefly luciferase gene into plasmid and transform Agrobacterium. *Grow Agrobacterium in large quantities and infect tobacco plant. http://cwx.prenhall.com/bookbind/pubbooks/horton3/medialib/media_portfolio/23.html

22. Yeast Artificial Chromosomes (YACs): Vectors that enable artificial chromosomes to be created and cloned into yeast. Features: Yeast telomere at each end. Yeast centromere sequence. Selectable marker (amino acid dependence, etc.) on each arm. Autonomously replicating sequence (ARS) for replication. Restriction sites (for DNA ligation). Useful for cloning very large DNA fragments up to 500 kb; useful for very large DNA fragments.

23. Bacterial Artificial Chromosomes (BACs): Vectors that enable artificial chromosomes to be created and cloned into E. coli. Features: • Useful for cloning up to 200 kb, but can be handled like regular bacterial plasmid vectors. • Useful for sequencing large stretches of chromosomal DNA; frequently used in genome sequencing projects. • Like other vectors, BACs contain: • Origin (ori) sequence derived from an E. coli plasmid called the F factor. • Multiple cloning sites (restriction sites). • Selectable markers (antibiotic resistance).

24. Fosmid: • Based on the E. coli bacterial F-plasmid. • Can insert 40 kb fragment of DNA. • Low copy number in the host (e.g., 1 fosmid). • Fosmids offer higher stability than comparable high copy number cosmids. Contain other features similar to plasmids/cosmids such as origin sequence and polylinker.

25. Recombinant DNA Libraries (2 major types): • Genomic/chromosomal library, Collection of cloned restriction enzyme digested DNAs containing at least one copy of every DNA sequence in a genome or chromosome. • Complementary DNA (cDNA) library, Collection of clones of DNA copies made from mRNA isolated from cells. • reverse transcriptase (RNA dependent DNA polymerase) • Synthesizes DNA from an RNA template • cDNA libraries reflect what is being expressed in cells. • # of clones required for a complete library can be calculated from the size of the genome and average size of overlapping fragments cut by restriction enzymes. • Library should contain many times more clones than the calculated minimum number of clones.

26. Genomic Library: 3 ways to make a genomic library: • Complete digestion (at all relevant restriction sites) • Produces a large number of short non-overlapping DNA clones. • Genes containing two or more restriction sites may be cloned in two or more pieces. • Mechanical shearing • Produces longer DNA fragments. • Ends are not uniform, requires enzymatic modification before fragments can be inserted into a cloning vector. • Partial digestion • Cut at a less frequent restriction site and limit the amount and time the enzyme is active. • Results in population of large overlapping fragments. • Fragments can be size selected by agarose electrophoresis. • Fragments have sticky ends and can be cloned directly.

28. Fig. 8.7, Partial digestion with Sau3A Results in a library of overlapping DNA fragments of various sizes.

29. Screening a genomic library (plasmid or cosmid): Plasmid vectors containing digested genomic DNA are transformed into E. coli and plated on selective medium (e.g., ampicillin). Colonies that grow are then are replicated onto a membrane (E. coli continues to grow on the membrane). Bacteria are lysed and DNA is denatured. Membrane bound DNA is next probed with complementary DNA (e.g., 32P radio-labeled DNA or flourescent probe). Complementary DNA in the probe is composed of DNA sequence you are looking for. Unbound probe DNA is washed off the filter. Hybridization of probed DNA is detected by exposure to X-ray film (or by chemiluminescence). Pattern is noted from exposure pattern of clones on X-ray film. Select clones that test positive and isolate for further analysis.

30. Screening a genomic library

31. In the case of a chromosomal library, screening can be reduced if target genes can be localized to a particular chromosome. Chromosomes can be separated by flow cytometry, a common method of fractionation. • Condensed chromosomes are stained with fluorescent dye. • Chromosomes separate based on the level of binding of the dye and are detected with a laser. http://www.ncbi.nlm.nih.gov/

32. cDNA Library: • cDNA is derived from mature mRNA, does not include introns. • cDNA may contain less information than the coding region. • cDNA library reflects gene activity of a cell at the time mRNAs are isolated (varies from tissue to tissue and with time). • mRNA degrades quickly after cell death, and typically requires immediate isolation (cryoprotectants can increase yield if immediate freezing is postponed by field work). • Creating a cDNA library: • Isolate mRNA • Synthesize cDNA • Clone cDNA

33. Creating a cDNA library - Step 1-Isolate mRNA: • Mature eukaryote mRNA has a poly-A tail at the 3’ end. • mRNA is isolated by passing cell lysate over a poly-T column composed of oligo dTs (deoxythymidylic acid) . • Poly-A tails stick to the oligo dTs and mRNAs are retained, all other molecules pass through the column.

34. http://www.ncbi.nlm.nih.gov/About/primer/images/Drip4.GIF

35. Creating a cDNA library - Step 2-cDNA synthesis: • Anneal a short oligo dT (TTTTTT) primer to the poly-A tail. • Primer is extended by reverse transcriptase 5’ to 3’ creating a mRNA-DNA hybrid. • mRNA is next degraded by Rnase H, but leaving small RNA fragments intact to be used as primers. • DNA polymerase I synthesizes new DNA 5’ to 3’ and removes the RNA primers. • DNA ligase connects the DNA fragments. • Result is a double-stranded cDNA copy of the mRNA.

36. Fig. 8.15, Synthesis of cDNA

37. Creating a cDNA library - Step 3 Cloning of cDNA: cDNA has blunt ends, thus need to add restriction site linkers or adapters to make them “sticky”. Use T4 DNA ligase and blunt end ligation to add restriction site linkers (or adapters) to each end of the cDNA. Next, digest the linkers with the same restriction enzyme used to cleave the vector. Mix cDNA with cut vector DNA in the presence of DNA ligase. Transform into an E. coli host for cloning. If cDNA has the same restriction site as the linkers, cDNA will be cloned in pieces. Solution, use adapters with single-stranded overhangs that match the restriction site on the vector.

38. Fig. 8.16, Cloning of cDNA using BamHI linkers Alternative: use adapter 5’-GATCCAGAC-3’ GTCTG-5’

39. Screening a cDNA library: cDNA libraries are used to detect or sequence genes for proteins (cDNAs are generated for genes that are transcribed!). If you know the DNA sequence for the protein coding gene you want to find, a homologous DNA probe can be used. If no homologous DNA sequence is available, cDNA can be probed with with an antibody that recognizes the protein. Expression vector: cloned cDNA is inserted into a vector between a promoter and transcription terminator before it is transformed. mRNA is transcribed from the cDNA and translated by E. coli. Colonies (now expressing proteins) are transferred to membrane. Membrane is incubated with labeled antibody probe that recognizes the protein. Colonies with bound antibodies leave a dark spot on X-ray film.

40. Fig. 10.4 - Transcribable vector containing a cDNA insert.

41. Fig. 10.5 Screening a cDNA library with antibody probe.

42. Screening a cDNA library (cont.): • Identified cDNA can be used to: • Isolate and sequence the gene for the protein (either with out without DNA sequence information). • Identify and compare homologous sequences in other types of organisms. • Quantify amount of mRNA synthesized from a gene and measure the level of gene expression. • Synthetic oligonucleotides might be used to probe the library if DNA sequences are available. • Complete cloned cDNA from other organisms showing a high level of homology also can be used as probes, heterologous probes.