Chapter 5: Sources of DNA: Genomic and CDNA Libraries

Genomic Library

Total gDNA from a single organism, stored in a population of identical vectors

  • Isolate pieces of genomic DNA (gene of interest)
  • Isolate fragments adjacent to a specific fragment
  • Gene mapping and genome sequencing
  • Promotor, introns, intergenic region, non-expressed genes, DNA → DNA


Collection of cloned cDNA fragments inserted into host cells, contain only mRNA sequence

  • Estimate transcript abundance
  • Identify novel genes, splice variants, amino acid sequence
  • Isolate coding regions for overexpression or to create fusional constructs
  • Expressed genes, transcript start site ORF, splice points, 5’cap 3’polyA tail, mRNA → cDNA

Creation of Genomic Library

Choose vector, fragment genome, ligate and transform, evaluate quality, store

Vector Choice

  • Goals, size of insert and genome, host compatible, ease of use
  • Often lambda because phage are easier to screen and transfer to membrane for hybridization, many viral particles in each plaque


genomic digests create fragments and create a ladder looking like a smear

  • Partial Digest: Incomplete digestion by limiting time, RE amount, temp, Mg conc.
  • Generates overlapping fragments to determine the order or make 2 diff libraries with different RE
  • Complete Digest: Non-overlapping and individual fragments is put into the library


precut vectors can be purchased, control ratio of insert to vector 3:1, to stop vector from taking 2 or more fragments, phosphatase plasmid vector to prevent self ligation, use highly competent cells


Transformation in bacteria, packaging extract in lambda, electroporation in yeast


Plate original transformation mix and count # of colonies to predict # of clones with inserts and determine complexity of library ( # of independent clones)


-80oC long term, cryoprotective agent (glycerol or DMSO) to prevent ice crystal formation denaturation and membrane damage, do not freeze and that freeze in aliquots

cDNA Library

  • Only 1.5% of genome encodes protein coding sequences, cannot clone RNA, no REs so uses copy DNA
  • Summary: Abundance of cDNA library reflects abundance of corresponding mRNA in the cells used, compare transcripts by comparing abundance and identity of libraries
  • Limitation: Only reflects specific tissue, difficult to recover long transcripts/rare transcripts/unspliced variants/non polyadenylated
  • Isolation: RNA containing extract produced by homogenizing tissue, inactivate RNase, flash freeze in buffer with phenol and salts to denature proteins and remove debris via centrifugation
  • Column Purification: Pour total extract on oligo-dT beads, bound mRNA eluted by lower salt & up temp
  • Made from total extract, oligonucleotide of dT used as primer for RT, RNA dependent DNA polymerase, synthesizes 5 to 3, to copy long strands use temp-resistant RT at high temp so RNA remains ss
  • Product is RNA-DNA complex: several techniques to replace RNA with DNA
  • Degrade RNA and inherent complementarity of DNA forms hairpin as primer
  • Nick the RNA with RNase H and use one of the RNAs as primer
  • Plasmids generally used to create cDNA libraries

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