BSL2016 / 2018 LEC 8 Genomic Libraries (1) What is a genomic library and why is it important? How does a genomic library differ from a cDNA library? cDNA LibraryGenomic Library Only contains expressed sequences mRNA/cDNA Contains the entire genome Does NOT contain tRNA or rRNA sequences tRNA and rRNA genes are Present. Does NOT contain non- coding sequences Contains EVERYTHING Promoters, introns, junk, Cannot be used for mappingEssential in genome Sequencing projects Cloned sequences are short typically 1-2 kb Cloned sequences are long up to 100kb

BSL2016 / 2018 LEC 8 Genomic Libraries (1) What is a genomic library and why is it important? A genomic library is a collection of cloned sequences which represents the entire genome. It allows the analysis of gene promoters which control how genes function (where and when they are expressed, and in response to which stimuli) It allows the creation of maps of the genome which detail which genes are located where, and the relative distance of one gene from the other. Used extensively in the Human Genome Sequencing Project.

BSL2016 / 2018 LEC 8 Genomic Libraries (1) How many clones are needed to ensure that the complete genome has been cloned? Depends on two factors: 1) Size of the genome; 2) Size of the cloned fragment Assuming that 17kb is the size of the cloned fragment…….. SpeciesGenome size ( bp ) No. of clones E. coli4 x D. melanogaster8 x x 10 4 H. sapiens3 x x 10 5 P. sativum4.5 x x 10 6 The larger the size of the cloned fragment, the smaller the number of clones.

BSL2016 / 2018 LEC 8 Genomic Libraries (1) What factors need to be taken into account when preparing genomic DNA for library construction? You need to cut DNA with restriction enzymes so that a series of overlapping genomic fragments is created : This is essential for gene isolation and for genome mapping

BSL2016 / 2018 LEC 8 Genomic Libraries (1) The restriction enzyme used to cleave the genomic DNA must NOT cut in the middle of genes, otherwise those genes will be “lost” from the library.

BSL2016 / 2018 LEC 8 Genomic Libraries (1) Which enzyme should you use to cleave genomic DNA? Remember that you want to clone long, overlapping fragments. All genes will be cleaved Complete cleavage > fragments too short Partial cleavage > fragments too long Fragments are too large to insert into most cloning vectors.

BSL2016 / 2018 LEC 8 Genomic Libraries (1) The best strategy to produce fragments which are long, and overlapping is to choose an enzyme (Sau3A) which cuts very frequently in the genome – not obvious!! But only to cleave a few of these sites at random (partial cleavage) Which will result in a population of long, randomly cleaved fragments.

BSL2016 / 2018 LEC 8 Genomic Libraries (1)

Bacteriophage lambda is the vector of choice for the creation of genomic libraries (apart from sequencing and mapping projects) Head (DNA) Tail

BSL2016 / 2018 LEC 8 Genomic Libraries (1) Phage genome is approx 50 kb long. In the process of head filling, a cos site is recognised, DNA is reeled in until the second cos site is reached, and is then cleaved. Central region of the genome is not essential for phage functions

BSL2016 / 2018 LEC 8 Genomic Libraries (1) These are called “replacement vectors” because the central non-essential (stuffer)region of the phage genome is replaced with genomicDNA

BSL2016 / 2018 LEC 8 Genomic Libraries (1) Lysis of the bacterial lawn results in “plaques”