The central Dogma DNA mRNA Protein

How does one isolate a gene for an inherited disorder There are three options Start with a candidate protein DNA Start with a candidate mRNA DNA Direct positional cloning DNA

Gene Libraries

Protein mRNA

Types of Gene libraries

A DNA library is a comprehensive collection of cloned DNA fragments which includes one with the gene of interest There are two types of libraries: Genomic libraries cDNA libraries

Library construction cDNA libraries and Genomic libraries

Tissue mRNA cDNA DNA Partially or completely digested DNA

cDNA libraries

Ligate DNA and vector

Genomic libraries

Introduce ligated vector/ target DNA into E.coli ( in vitro packaging or transformation) Titer and characterize library

Screen for desired clones

Amplify for long term storage

Construction of Genomic libraries

GENOMIC LIBRARIES Creating a genomic library consists of digesting the entire genome of a cell with a restriction enzyme in order to produce a large number of DNA fragments Some fragments will contain genes, others will contain portions of genes and still others will contain noncoding DNA
Target DNA

Digest with restriction enzyme

DNA recombination, packaging ,assembly

LIBRARY DISADVANTAGES cDNA LIBRARIES Genomic libraries have a large number of fragments that contain non-coding DNA, which makes this less than ideal. It does, however, represent all DNA sequences of an organism. A different cDNA library must be made for each cell type, since the library is based on mRNA and contains only genome that has been transcribed.

Creating a cDNA library consists of isolating mRNA from a cell, then using reverse transcriptase to make complementary DNA (cDNA). DNA polymerase is then used to make the cDNA double-stranded. It is presumed that mRNA corresponds to genes

RESTRICTION ENDONUCLEASES Restriction enzymes Cut DNA into specific fragments Recognize specific base sequences in double stranded DNA and cleave both strands of the duplex at specific places Characteristics of restriction enzymes Cut DNA sequence specifically Bacterial enzymes : many purified and available commercially Restriction modification system -Bacteria have enzymes that will cleave foreign DNA hence restrict the entry of viral DNA . -To prevent the bacteria s own DNA from being cut there is a second enzyme that methylates the same site recognized by the restriction enzymes (modifies the site) Named e.g. Eco R1 for the bacterial genus, species, strain and type Recognize specific 4-8 bp sequences -sequences have symmetry (they are palindromes) -the cut ends are either Blunt Staggered (overhangs) cohesive ends facilities cloning the DNA Frequency of cutting 4 base cutter 4 4 = 256bp, 5 base cutter 4 5 = 1024bp , 6 base cutter 4 6 = 4,096bp 8 base cutter 4 8 = 65,536bp

Products generated by restriction enzymes

Formation of recombinant DNA molecules

Recombinant DNA

Isolation of ~20kb fragments provide optimally sized DNAs for cloning in bacteriophage Partial digestion with a frequent cutter (4-base cutter) allows production of overlapping fragments since not every site is cut Overlapping fragments insures that all sequences in the genome are cloned Overlapping fragments allows larger physical maps to be constructed as contiguous chromosomal regions (contigs) are put together from the sequence data.

GENOMIC LIBRARY CONSTRUCTION Construction of a genomic library Disadvantage of genomic library The Eukaryotes have intron sequence
EcoR1

Vector DNA (bacteriophage lambda) Lambda has a linear double stranded DNA genome The left and the right arms are essential for the phage replication cycle The internal dispensable fragment is

Internal fragment (dispensable for phage growth)

Advantage of genomic library The regulatory sequences flanking the coding portion of the gene The non coding intervening sequences The various members of a multigene family which often lies close together in the genome The evolution of DNA sequences, including duplication and rearrangement as seen in comparasion of the DNA of different species The interspersion of transposable elements

HOW MANY COLONIES HAVE TO BE SCREENED This is decided by the size of the cloned fragment and the size of the genome A simple statistics based on the Poisson distribution specifics the number of independent clones N that must be screened to isolate a particular sequence with probability P given by N= In(1-P)/In(1-(I/G) I = the size of the average cloned fragment G= the size of the target genome in base pairs For a 95% chances of isolating an individual sequence from a typical genome using a phage lambda vector eg., genome size 2.8 x 106 /20 = 1.4x 105 N= In (1-0.95)/ In[1-(1/1.4x 105)] = 4.2 x 105

cDNA LIBRARIES

mRNA has a unique feature in the poly A tail, allowing easy isolation of mRNA from total cellular RNA (rRNA and tRNA) The poly A tail readily binds to oligo (dT) cellulose which can be made into a column. By passing total RNA over the column, mRNA is retained while the rest passes through

cDNA LIBRARIES
In order to synthesize double-stranded DNA, RNase H must first be used RNase H is derived from E. coli and recognizes RNA-DNA hybrids It digests the RNA into small fragments E. coli DNA polymerase I uses the original cDNA template to synthesize the complementary strand,replacing the RNA except for a small piece at the 5 end The DNA is made continuous by ligating with DNA ligase Its also possible to attach artificial sites to the cDNA ends
CONSTRUCTION of cDNA LIBRARY A cDNA library is a collection of all the expressed DNA of a particular cell type or tissue. Eg., A cDNA library from the bone marrow cells will have many clones with the cDNA for hemoglobin alpha and beta chains. The cDNA libraries are advantageous since they don't have the intron sequences which is present in the genomic libraries cDNA clones find application where bacterial expression of the foreign DNA is necessary, either as a prerequisite for detecting the clone or because the polypeptide product is the primary objective cDNA cloning can be used to study the temporally regulated gene expression in development, or tissue specific

CONSTRUCTION of cDNA LIBRARY

CONSTRUCTION of cDNA LIBRARY (continued)

Double stranded cDNA copies of mRNA with Eco R1 cohesive ends are ready to ligate into a bacteriophage lambda vector cut with Eco R1

cDNA: first strand synthesis

mRNA 5 Anneal oligo-dT primer Reverse transcriptase: RNA-directed DNA polymerase RNase H Hydrolyze remaining RNA with base TTTTT 5 Product is complementary DNA, called cDNA. It is equivalent to the template strand of the duplex DNA. TTTTT AAAAAAA 3 TTTTT 5 dNTPs AAAAAAA 3 TTTTT 5 AAAAAAA 3

cDNA: second strand synthesis

cDNA Terminal deoxynucleotidyl transferase CCCC Oligo dG primer & oligo-dT primer 5 3 5 3 GGGG CCCC Taq DNA polymerase GGGG CCCC Duplex cDNA TTTTT 5 dNTPs PCR AAAAA 3 TTTTT 5

TTTTT 5 dCTPs TTTTT 5

Ligate duplex cDNA into a plasmid

Duplex cDNA 5 GGGG CCCC 3 Cut the adaptor GGGG CCCC Ligate duplex cDNA into a plasmid AAAAA TTTTT AAAAA 3 TTTTT 5

SCREENING OF RECOMBINANT CLONE

Bacteriophage or plasmid libraries

Plate Library

Screen library by Hybridization to oligonucleotide probes Colony hybridization Immunochemical method Transform the population of cDNA plasmids into bacteria. Result is a cDNA library. Link to animation pictures http://www.rvc.ac.uk/Extranet/DNA_1/2_insertion.htm Insertional inactivation

Labeling of DNA probes using random hexamers

Double stranded DNA Denature by heating

Labeling of DNA probes using random hexamers: Peroxidase labelling

Double stranded DNA Denature by heating

Hybridize with random hexamer 3NNNNNN5 Add Klenow DNA polumerase dNTP and labelled dNTP
Label: Radioactive isotopes (P32) Biotin, digoxigenin, enzymes

Add Horse radish peoxidase + gluteraldehyde

Horse radish peoxidase labelled DNA

Screening of recombinants: Colony hybridization

LB agar

Colony hybridization

cont..

LB agar

Transfer colonies on nylon membrane

Lyse and denature DNA

Probe with labelled DNA

Hybridization

Stringency wash

Autoradiograph

Positive