BCHM 4116 1st Edition Lecture 8 Outline of Last Lecture I Vectors and Molecular Cloning II Alpha complementation III Strategies to increase number of clones Outline of Current Lecture IV Gibson Assembly V Gateway Cloning Recombination based cloning VI DNA library Construction VII Genomic DNA library construction Phage vector human DNA VIII Making a cDNA library Current Lecture Gibson Assembly There are three parts to this DNA preparation Gibson assembly and transformation DNA Preparation I Require a linear vector II Requires DNA inserts with overlapping ends Gibson Assembly I An exonuclease chews back 5 ends to create single stranded 3 overhangs These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute II DNA polymerase fills in gaps within each annealed fragment III DNA ligase seals nicks in the assembled DNA Advantages of Gibson Assembly include being able to do dozens of molecules at a time Transformation I This is when the completed vector is mixed with bacteria that can then uptake the vector and start transcription translation Overview of Gibson Assembly Gateway Cloning Recombination based cloning Step A is used to get the gene of interest into a vector The gene is inserted into the donor vector and the final product is called the entry clone The donor vector has a selectable marker that gives it resistance to a certain antibiotic When grown on a plate with this antibiotic only the bacteria that have the insert of choice and the selectable marker donor vector will survive This entry clone however cannot express the genes as proteins because it doesn t have all the promoter sites it needs Step B takes the product on step A and mixes it with a destination vector that has a different selectable marker Through recombination the gene of choice is transferred to the destination vector This destination vector is called the expression clone as the finished product This expression clone has the promotors to express proteins for bacteria or mammalian expression systems DNA Library Construction A DNA library is a set of individual units or clones that represents the total sequences from a particular source Libraries can originate from I The entire genome of an organism a genomic library II The total expressed sequences mRNA in a sample a cDNA library Whatever the source the DNA inserts are introduced into a vector for propagation in E coli or other hosts Genomic library Contains DNA sequences from the total genome of an organism but expressed sequences and non expressed untranscribed sequences DNA is identical for essentially all cells so the source of DNA is unimportant all genes present in all cells Contains intergenic regions and signals for proper eukaryotic gene expression i e promoters enhancers terminators introns Genomic DNA library construction Phage vector human DNA 1 Phage bacteria is cut with Bam H1 into two pieces left and right arm 2 The internal fragment is discarded 3 Human genomic DNA is cut up into 20 kb fragment using Sau 3A 4 base cutter 4 Sau 3A forms overhangs that are compatible with Bam H1 5 The human genomic DNA fragments are then inserted between the left and right arms of the phage DNA Making a cDNA library 1 Add a solution of total RNA in a column with salt The column has a cellulose matrix that has oligo dT chains attached 2 The column is washed with salt solution to remove any non mRNA material such as rRNA and tRNA Eukaryotic mRNA has a poly A tail that hybridizes with oligo dT chains on the cellulose whereas rRNA and tRNA pass right through the column 3 The mRNA bound to column with oligo dT is eluted by adding water Low salt concentration destabilizes DNA allowing the collection and evaluation of the mRNA eluted from the column A visual of the process How is cDNA made mRNA isolated from the column is mixed with oligo dT primers which anneals to the poly A tail of the mRNA Then reverse transcriptase is added along with substrates dATP dTTP dGTP dCTP This synthesizes the first strand of cDNA from the mRNA template Then RNase H is added to the mix RNase H is a endonuclease which recognizes the mRNA cDNA duplex and cuts the mRNA leaving behind large gaps in the sequence DNA polymerase then fills in the gaps which are then ligated together by ligase This is then the cDNA duplex Overhanging ends are added at the end to aid in cloning Here is a visual