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

cDNA

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

Fragmentation

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

Ligation

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

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

Evaluate

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

Storage

-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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