Isolating genes

We talked about getting the gene for the insulin hormone, putting it into a plasmid which we put into bacteria, and getting the bacteria to make our insulin.  How do we get the insulin gene in the first place?

Libraries

-          somehow cut up genome from which you are trying to isolate a gene (in this case the human genome) and put the pieces into a vector

o       makes a storable collection of clones that contains copies of every sequence in the whole genome

-          how to cut up?

o       Can use restriction enzymes

o       Can just shear DNA to make random pieces

-          vectors (Table 9.2)

o       plasmids

§         small inserts (say 5kb or less)

§         can have about 200 bacterial colonies on a plate that are well separated and don’t grow into each other

o       phage lambda (l)

§         phage is a virus that infect bacteria

§         dsDNA genome

§         can insert up to 25kb of dsDNA

§         Can easily have 1000 separate lambda/plate

o       Cosmid

§         Derived from phage lambda

§         Is maintained like a plasmid in a bacterial cell

§         Can insert up to 45kb

o       Phage P1

§         Derived from a phage

§         Can insert up to 100kb

o       BAC

§         Bacterial artificial chromosome

§         A human-made chromosome, circular, contains chromosomal origin or replication

§         Can insert up to 150kb

o       YAC

§         Yeast artificial chromosome

§         A human-made chromosome, linear

§         Can insert up to 2 megabases, but often several hundred kb

Why is size of insert such an important consideration in vectors?

-          if making human genomic library, human genome is 3X10⁹ bp

o       Plasmid say 5kb/plasmid insert

§         3X10⁹ (size of genome)/5X10³ (av. size of insert) = 6X10⁵ clones

§         = 3000 plates if 200 colonies/plate

o       Phage say 20kb/phage insert

§         3X10⁹/2X10⁴ = 1.5X10⁵ clones

§         = 150 plates if 1000 phage/plate

o       BAC av size of 100kb insert

§         3X10⁹/1X10⁵ = 3000 clones

§         = 15 plates is 200 colonies/plate

What about genome equivalents?

-          we are assuming in the example above that the genome was cut up into completely nonoverlapping fragments and that each vector will take one fragment (one genome equivalent)

-          in reality you are cutting up many genomes and vectors are getting the same or overlapping fragments

o       so if you only had 3000 clones in the BAC example above, it is very likely that some sequences would be present multiple times, and some not at all

o       so need to make a bigger library, like to have four or five genome equivalents

§         on average each gene present 4 or 5 times in library

§         95% chance that there is at least one of every gene

cDNA libraries

-          when insert is not genomic DNA but rather cDNA

-          cDNA is a dsDNA copy of a mRNA

o       construction (Fig. 9.10)

§         isolate mRNA from a tissue

§         use enzyme reverse transcriptase and TTTTTTT primer to make a DNA copy of the mRNA

§         destroy RNA with RNAse enzyme

§         take advantage of hairpins at 3’ end to prime complementary strand synthesis

§         end you get a double stranded DNA copy of an mRNA

-          eliminates problem of introns in genomic DNA sequence

-          also allows you to make library of only genes expressed by a particular cell type or under a particular condition

o       this can be an advantage or a disadvantage (see Fig. 9.11)