Types of cloning vectors 1. plasmids (about 20kb)

1. Types of cloning vectors 1. plasmids (about 20kb) 2. Bacteriophage (bacterial viruses), 30-50kb inserts 3. Cosmids (35-50kb insert) 4. BACs • Use fertility F plasmid • 75-300kb inserts possible • developed during the human genome project 5. YACs • Mimics yeast chromosome • Contains all regions for replication (yeast ori and centromere) • 100-1000kb inserts poss. • developed during the human genome project

2. Viral vectors 1. Bacteriophage Lambda • Virus of E.coli • Lytic or Lysogenic life style • Virulent phage • do not integrate their DNA into the host bacterium genome and they usually kill the host (lytic) • Temperate phage may integrate into the host genome causing lysogeny

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6. Bacteriophage lambda vectors are commonly used for construction of genomic libraries Packaging of bacteriophage lambda Packaging of DNA into the head does not require a full length of wild type DNA. Length of between 78-105% can be packaged (37-53kb) • To get packaging need 2 cos ends one at each end. • Usually 50 kb apart and this ensures only 50kb gets packaged • If further than 53kb , no packaging • An enzyme at entrance to head recognises and cuts at cos site

8. During replication, the phage DNA is produced in a concatameric form, which is cleaved by appropriate endonucleases to allow packaging of a single genome within the phage capsid. • It was found that internal regions of the phage genome, which were not essential to phage replication, could be removed and replaced with DNA of interest. • This hybrid DNA could be efficiently packaged, and form an infective phage. Advantages of this type of system vs plasmids like pBR322 • The phage genome is able to package efficiently with DNA inserts as large as 20 Kb. • the packaged phage are highly infectious and infect E. coli at a much higher efficiency than plasmid transformation methods.

9. Bacteriophage Lambda vectors • Derivatives have been manipulated to have unique restriction enzyme sites • Some are insertion some are replacement vectors • Some have replacement areas flanked by cutting sites • 40% of the wild type lambda is dispensible and can be replaced e.g. of lambda cloning vector (see diagram) • lambda sequences have been devised with 2 Bam H sites flanking the insertion excision region • When cut with Bam get 3 segments • Left arm has info for heads and tails • Right arm has info for DNA replication and lysis • Middle fragment can be replaced with cloned DNA about 20kb long.

10. replacement vector replacement vectors replacement vectors

11. Insertion Vector -Can not fit as much new foreign DNA in

13. In vitro packaging systems • Can be made • Purified empty heads, lambda DNA with foreign insert (50kb with cos ends), and tail assemblies in test tube. • Result in infective bacteriophage • Uses • Cloning pieces too big for plasmids • Generation of ds DNA

14. M13 , a single stranded filamentous phage • Phage DNA is packaged in the core of a helical particle • The length of particle is dependant on length of DNA • In all M13 preps the following occur • polyphage- more than one genome length • minphage- 0.2-0.5 genome length • maxiphage- genetically defective but more than one genome length • Molecular biologists use this to create cloning vectors • Can insert long stretches of DNA into non essential regions • No sharp cut off in length that can be packaged • Some decrease in efficiency of packaging with increasing length • 10% longer not affected, 50% longer replicate more slowly

15. How M13 infects and reproduces • infects through pili • Protein coat is stripped and ss DNA is converted to double stranded replicative form • DNA relicated by “rolling circle method” • New particles assembled • 200 particles per infected cell per generation • M13 released without lysis • No lysis on bacterial lawn, generally do in liquid culture. Uses • Cloning • Ss DNA for probes, sequencing • Phage display technology, M13 will produce foreign protein on surface as part of its protein coat, can use to generate specific antibodies

16. Adeno virus • Human virus • Used to insert DNA into human cells • Gene therapy • Normally causes respiratory diseases and pink eye. Want virus to infect our cells but not cause illness

18. Cosmids • Cross between phage and plasmid • Circular ds DNA • Contain cos sites and can be packaged into phage heads • Carry more DNA than plasmid and can be maintained and manipulated as plasmids Cloning using cosmids • Clone into vector as would with plasmid • Introduce DNA to cell as you would with phage • Propagate as you would a plasmid • Cosmids have about 5kb DNA and therefore can get ~ 33-48kb foreign DNA into a phage head.

19. Measurement of viral growth • Must grow virus on host cells to see anything. Can't grow virus without cells. • To quantify viruses, need some way to get flat surface of growing cells, allow virus-infected cells to spread radially where present = plaque. • In bacterial cells this is easy. Spread "lawn" of bacteria on plate, add diluted phage suspension or culture infected with phages. After 6-8 hours can see plaques in E. coli.

20. Bacteria have three mechanisms to accomplish genetic recombination (mixing up of DNA): 1. transformation -acquire new genes by taking up DNA molecules from their surroundings 2. conjugation -can transfer a portion of their chromosome to a recipient with which they are in direct contact usually via a plasmid 3. transduction- via Bacteriophages - in the process of assembling new virus particles, some host DNA may be incorporated in them

21. Bacterial artificial chromosomes (BAC): The F factor plasmid has the ability to continue to function even when integrated into a complete bacterial chromosome. Highly modified F plasmids have been generated that are capable of cloning very large inserts of up to 300,000 base pairs. One feature is the incorporation of cut sites for restriction endonucleases with eight base cut sites. Such endonucleases cut DNA less frequently and thus generate larger fragments for cloning. Bacterial artificial chromosomes are sometimes introduced into their host cells by electroporation, which consists of a brief treatment with high voltage electric current that momentarily disrupts the cell membranes and facilitates entry of large DNA molecules. Once in the cell, the BAC replicate like F plasmids. Yeast Artificial Chromosomes (YAC): a yeast artificial chromosome (YAC) contains a yeast origin of replication a centromere, a telomere at each end a large inserted DNA sequence of up to about 500 kb Prior to insertion of the foreign DNA, the essential components of the YAC are maintained in bacterial cells as circular plasmids.