Genomic and cDNA library (Part I) - Genomics - Biology PDF

Genomic and cDNA library (Part I)  Introduction The use of genetic information is a powerful tool that today is becoming more readily available to scientists. In order to use this powerful tool it necessary to know how to navigate throughout the entire gen...

Genomic and cDNA library (Part I)  Introduction The use of genetic information is a powerful tool that today is becoming more readily available to scientists. In order to use this powerful tool it necessary to know how to navigate throughout the entire genome. The human genome is about 3.3 x 109 Kbp. In humans this project is known as Human Genome Project.  How to generate Genomic information Tissue/ Cell Vectors (plasmids, phage, Genomic information is obtained through: Cosmid, BAC, YAC etc. 1. A genomic library mRNA DNA. Genomic library is a collection of cloned sequences which represents the entire cDNA Fractionation genome (exons and introns). (methylation, (partial or. It allows the analysis of gene promoters linker complete which control how genes function (where and addition) digestion) when they are expressed, and in response to Restriction which stimuli). It allows the creation of maps of the genome which detail which genes are Ligation located where, and the relative distance of one gene from the other. Transformation, in vitro packaging etc. Used extensively in the Human Genome Sequencing Project. Screening for positive clone 2. Complementary DNA (cDNA) library that contains only expressed genomic information (only exonic sequences) Amplification for long term storage  Tools needed to generate genomic and cDNA libraries Enzymes: Restriction enzymes DNA polymerase I Reverse transcriptase Terminal transferase DNA ligase DNA methylase Vectors: Plasmids Bactriophage Lambda (l phage) Yeast Artificial Chromosome (YAC) Bacterial Artificial Chromosome (BAC)  Restriction Enzymes Recognize a specific target sequence (restriction sites) in DNA, and then break the DNA (both strands), within or close to, the recognition site. e.g. EcoRI, PstI and SmaI. Usually require Mg2+  Some generate 5' overhangs - eg: EcoRI Some generate 3' overhangs - eg: PstI Some generate blunt ends - eg: SmaI  Restriction endonucleases are a natural part of the bacterial defense system Part of the restriction/modification system found in many bacteria These enzymes RESTRICT the ability of foreign DNA (such as bacteriophage DNA) to infect/invade the host bacterial cell by cutting it up (degrading it) The host DNA is MODIFIED by METHYLATION of the sequences these enzymes recognize. Methyl groups are added to C or A nucleotides in order to protect the bacterial host DNA from degradation by its own enzymes Reverse transcriptase DNA primer DNA-dependent DNA polymerase  Terminal Transferase Terminal transferase catalyzes the addition of deoxynucleotides to the 3' hydroxyl terminus of DNA molecules. It is a template independent DNA polymerase. Useful for adding homopolymeric tails or single nucleotides to the 3' ends of DNA strands. DNA ligase  Vectors Vector: vehicle used to transfer genetic material to a target cell Characterics of vectors ： Origin of replication Selection marker Multiple cloning site Capacity  Plasmid A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae Features of Plasmid used in molecular biology:. Relatively small molecular weight. Drug resistance gene. Multiple cloning site. Origin site for replication Application of plasmids:. cloning. Sequencing. In vitro transcription. Gene expression to produce protein in bacteria  λ-phage as a Vector - λbacteriophage The virus particle consists of a head and a tail that can have tail fibres. - The head contains 48,490 base pairs of double- stranded, linear DNA flanked by 12-base-pair, single- Replaceable stranded segments that make up the two strands of the cos site. region - In its circular form in the host cytoplasm, the phage genome therefore is 48,502 base pairs in length. - The prophage exists as a linear section of DNA inserted into the host chromosome - Lytic or lysogenic - Cloning capacity: 23 Kbp insert Lytic vs. lysogenic cycle of the λ-phage Molecular Events leading to the lysogenic cycle Virus circularization Recombination and integration into the bactrial chromosome  Yeast Artificial Chromosome (YAC) vector has yeast sequence for: 1. Chromosomal DNA replication. TEL: segment for telomeric DNA sequence. CEN 4: centromere sequence. ARS: autonomously repliucationg sequence (similar to origin of replication) 2. Selection of recombinants. TRP1, URA 3: yesat selectable markerks. SUP 4: The yeast SUP4 gene contains a cloning site used as a colour marker for selection of YACs containing exogenous insert DNA. SUP 4 causes the accumulation of red pigment. The host cells are normally red, and those transformed with YAC only, will form colorless colonies. ng capacity: 0.5-2 Mbp es carrying recombinants YAC are propagated in Saccharomyces cerevisiae  Bacterial Artificial Chromosome (YAC) - BAC vectors are similar to standard E. coli plasmid vectors. - Contain the origin of replication and genes encoding the ORI-binding proteins required for plasmid replication - Low copy number (1-2 copies per cell) - Transformation by electroporation of recombinant DNA into E. coli - Can accommodate DNA fragments up to 300 Kbp