week 9_genomic libraries posted

8/3/2019 Week 9_Genomic Libraries Posted

1/34

BI 312 Week 9 Lecture Overview

DNA libraries

Creation

Uses

Genomic vs. cDNA


2/34

If I want to clone MY FAVOURITE

GENE, how do I get the DNA in the

first place??

PCR

Order it!

Request it from another scientistScreen a genetic library

Some of the following gene library slides were taken from Dr. Mankins lecture found online at http://www.uic.edu/classes/phar/phar331/lecture7/
http://www.uic.edu/classes/phar/phar331/lecture7/http://www.uic.edu/classes/phar/phar331/lecture7/


3/34

GENE LIBRARIES

How do you clone one

particular gene from

thousands of genes present

in the genome?

Genome Sizes

E. coli 4.6 million basepairsHousefly 900 million basepairs

Human 3.4 billion basepairs

Pine tree 68 billion basepairs

NOT ASIMPLE

TASK!!!


4/34

The Strategy

Clone everything,then find what you

actually need


5/34

Creating a Genomic Library

1. Isolate chromosomal DNA(Remember you can useANY

tissue source--chromosomal DNAis the same in every cell in an

organism)

2. Digest with a restriction enzyme

3. Digest vector with the samerestriction enzyme

4. Ligate the fragments of

chromosomal DNA with the vector.


6/34

Making a representative library

In order to guarantee that youll be able to find yourgene in the library, you need:

the entire genome to be cloned into the vectors each region of the genome represented at least 2 or 3 times


7/34

Preparing the genomic DNA: partial

digests Time / amount of enzyme in digest is limited so

DNA is NOT cut at every recognition sequence

This will produce longer fragments This will hopefully allow you to clone the full-

length region you desire, and you will requirefewer recombinant clones to cover the library


8/34


9/34

Constructing the Library

ligate fragments intovectors

transform ligation

reaction intocompetent cells plate on selective

media only cells that

received arecombinant vectorwill grow; others willbe killed by theselection process


10/34

Constructing the Library

Plate transformed cells on

to agar + antibiotic

Each transformed cell thathas a plasmid will form a

colony; all cells in the

colony have the sameplasmid and cells in

different colonies havedifferent plasmids.


11/34

Choosing your vector the vector you use is determined by the size of the

genome you are working with

prokaryotes have smaller genomes so it is possible tomake a genomic library using a plasmid vector

genes are shorter and inserts can be smaller (5-10 kb) a representative library can be made from a few thousand

recombinants

eukaryotes have larger genomes and require vectors

capable of holding larger inserts genes and inserts are larger, vectors need to hold much larger

fragments of DNA (~20kb)

can use phage, BAC, PAC or YAC


12/34

Making a Phage Library

Fig 7-12 Lodish et al, 4th ed

mix recombinantphages with E. coli

host plate in soft-top agar

isolate phage DNA

from plaques Sometimes 2 different

RE enzymes are used


13/34

Why 2 different RE enzymes ? One of these enzymes must cut the genome frequently (ie a 4-mer; Sau3A) so

that we can obtain a random population of genomic fragments (lots offragments), while the other must cut uniquely in the vector (ie a 6-merenzyme;BamHI) to give specificity(so that we get the gene we are looking for).

SauIIIA and BamHI are the most common enzymes used for this purpose.

BamHI

Cuts vector

SauIIIA

Cuts genomicDNA

GATC

CTAG

GATC

CTAG

G GATCC

CCTAG GG GATCC

CCTAG G


14/34

Cut genomic DNA with SauIIIA

Cut DNA with BamHI

SauIIIA

Cuts genomic

DNA

GATC

CTAG

GATC

CTAG

BamHI

Cuts vector G GATCC

CCTAG G

G GATCC

CCTAG G

Remove replaceable region


15/34

Mix target DNA fragments + arms

Target DNAfragments

CCTAG GGGATC GATCC

CTAG

GATC

CTAG

G

CCTAGGATCC

G

RecombinantDNA

+

arms


16/34

Genomic Libraries: Review

1.

2.

3.4.

5.


17/34

Screening Genomic Libraries:

finding your gene Need to determine which colony is made

up of cells that contain a plasmid with

your gene inserted 4 common methods:

Phenotypic screening

Protein Activity

Immunological screening

DNA hybridization


18/34

Phenotypic Screening

Simplest method

Used when the clonedgene encodes a protein

that changes someproperty of the cells inan obvious and readilyvisible way

For example, the proteinencoded by the clonedgene changes the colourof the transformed cells


19/34

Protein Activity

Used when target gene encodes a proteinthat is an enzyme not native to the host

cells, and that enzyme activity can bereadily detected by eye

Replica-plate on to media that contains

the substrate for the enzyme; onlycolonies containing your gene will act onthe substrate and produce visible products


20/34

Replica-plating

that contains substrate forbiochemical reaction


21/34

Replica-plating

To transfer colonies from one plate toanother maintaining their exact locations,relative to each other and the plate.

Can be accomplished using a sterile fabricpad or nylon filter

Selective/differential media


22/34

Immunological Screening

Using antibodies to screen

the transformed cells Transfer (lift) colonies to a

nylon filter Some of the protein will be

transferred to the filter Hybridize filter with

antibodies that recognizethe protein encoded byyour gene

Positive colonies producecolour

Used when gene of interest is transcribed andtranslated in to protein


23/34

Screening by DNA Hybridization

Requires that the sequence of the target gene isknown

Target DNA (recombinants) is denatured andimmobilized

Design single-stranded probe complementary totarget

Label probe so that only colonies containing thetarget gene light up

Can use radioactivity or colourimetric-basedlabelling strategy


24/34


Glick & Pasternak, Fig 2.17


25/34



26/34



27/34

Screening a Phage Library by DNA

hybridization

Fig. 7-18, Lodish et al. (4th ed.)


28/34


in general, probes need to be 15-20 nt long in order toguarantee that they are unique (i.e. will bind ONLY to your

gene of interest)


29/34

The Challenge with Eukaryotes Eukaryotes have introns which are removed in the

nucleus after transcription but before translation.

Introns can be longer than exons (as much as 90% of agene!); single gene can be very spread out in thegenome and hard to isolate / analyze

Intron-containing genes cannot be expresed (i.e. madein to proteins) in bacterial cells because prokaryotes

dont have splicing machinery to remove introns


30/34

cDNA

cDNA = complementary DNA Generated from mRNA (so introns have been removed)

using an enzyme called Reverse Transcriptase; makesDNA from an RNA template


31/34

Generating cDNA: first isolate

mRNA Isolate total RNA from eukaryotic

cells

Translated mRNAs contain a

stretch of adenine residues at their3 end (poly A tail)

Use this feature to purify themRNA from the more abundanttRNA and rRNA: bind mRNA tosolid matrix containing shortstretches of thymidine residues(olido dT column)

Lodish et al; Fig 7-14


32/34

Generating a cDNA Library

ds cDNA can thenbe cloned in to avector, andtransformed intoan appropriatehost

cDNA libraries canbe useful for

studying tissue-specific expressionof mRNAs


33/34

Genomic vs. cDNA Libraries

Genomic Source is chromosomal DNA

Include introns

Can be made from ANY tissue source (chromosomalDNA is identical in all cells)

cDNA Source is mRNA

Contains protein-coding sequence (no introns; noregulatory elements associated with genes (e.ipromoters)

Each tissue source gives a different library since

specific mRNAs are expressed in each tissue


34/34

Choosing Your Library

What you want to do with your favourite geneonce you clone it will determine the type oflibrary you should use

Ask the following questions. Do you want tostudy: fragments of the complete genome? DNA sequence?

promoter regions? introns? intron/exon boundaries? protein expression?

week 9_genomic libraries posted

Documents