Uses of genomic library
- architecture and structure of genes
- sequencing of entire genome
- control of expression
Definition of DNA library
A collection of all DNA sequences that represent the genome of an organism/cell, each of which has been cloned into a vector to enable production, isolation, storage and analysis
Uses of cDNA libraries
- sequence of protein coding region
- production of new or modified protein
- gene expression: tissue specific and temporal pattern
How big should a genomic library be?
Must contain sufficient number of recombinants in order to ensure a high probability of it containing any particular sequence of genome
Process of constructing a genomic library
- purify genomic DNA
- fragment the DNA (restriction enzymes)
- clone the fragments into vectors
Why use partial digestion in genomic libraries?
- overlapping sequences allow for ‘chromosome walking’
- can use the end of one insert as a DNA probe to screen the library for adjacent fragments
Methods for having overlapping clone s
- partial digestion with restriction enzyme
- mechanical shearing
How to achieve a partial digestion of genomic DNA
- vary the amount of enzyme
- increase the time of digestion
- reduce the temp of digestion
How is partially digested genomic DNA fractionated?
By running the digested DNA on a sucrose gradient
Two ways that a lambda phage can carry DNA
- insertion vector (small 7kb)
- replacement vector (large 20kb)
Advantage of using lambda phage
Enables infection (a more efficient way of introducing DNA in bacteria)
What are cosmids?
Combo of a plasmid and a phage
Methods of screening gnomic libraries
- hybridisation
- colony and plaque hybridisation
How to isolate mRNA
- most mRNAs are polyadenylated at their 3’ends
- oligo dt can be bound to the polyA tail and used to recover the mRNA
How to screen cDNA libraries
Use nucleic acid probe to screen the library based on hybridization with nucleic acids
How to screen for cDNA protein product
- use expression vector
- introduce cDNA in the frame
- down stream to B-gal
- will be expressed during the lysogenc cycle of the virus
What is RACE used for?
To obtain the full-length sequence of an RNA transcript found within a cell
How do marathon libraries for PCR based cloning work?
- need knowledge of a short DNA sequence of the gene to be cloned
- obtain dsDNA and ligase adaptors to the ends
- these adaptors are complimentary and can perform PCR to synthesise fragment corresponding to desired gene
Steps in creating a cDNA library
- mRNA isolation/purification
- check RNA integrity
- fracionate and enrich RNA
- synthesis of cDNA
- treatment of cDNA
- ligate to vector
What is needed for cDNA creation?
- reverse transcriptase
- dNTPs
- primers
Characteristics of lambda phage
- dsDNA around 50kb
- has lytic and lysogenc life cycles
- central 20kb not required for the lytic cycle
- replication and packing requires 50kb DNA and 10kb
- packaging requires recognition sequences 50kb apart
Characteristics of a cosmic
- has COS site
- packages like a phage
- once in bacteria, acts like a plasmid
Definition of screening
Process of identifying one particular clone, containing a gene of interest
Process of hybridisation
- transfer DNA onto nylon or nitrocellulose gel
- hybridize with probe
- remove un hybridized probe
- visualize (xray)
- select positive from master plate
