© 2006 jones and bartlett publishers chapter 10 recombinant dna techniques 10.1cloning dna- basics...

© 2006 Jones and Bartlett Publishers

Chapter 10

Recombinant DNA Techniques10.1 cloning DNA- basics10.4 transgenic organisms - reverse genetics10.5 genetic engineering

10.1 Recombinant DNA Techniques

cut DNA with restriction enzymetake fragments

reassemble in new combinationsput back into organism (cell)

transgenic organism

(gene cloning)


restriction enzymes

(gene cloning)

cut DNA at specific sequencesrestriction sites(palindromes)


restriction enzymes

(gene cloning)

sticky ends5’ overhang3’ overhang

(complementary)blunt ends


Fig. 10.2. Two types of cuts made by restriction enzymes


restriction enzymes

(gene cloning)

5’-----GAATTC-----3’3’-----CTTAAG-----5’

3’ 5’5’-----GAATT C-----3’3’-----C TTAAG-----5’

5’ 3’

EcoRI

stick

y ends


restriction enzymes

(gene cloning)

5’-----GAATTC-----3’3’-----CTTAAG-----5’

3 5’5’-----GAATTC-----3’3’-----CTTAAG-----5’

EcoRI

DNA ligase3’5’

5’3’


Fig. 10.1. Circularization of DNA fragments produced by a restriction enzyme


restriction enzymes

(gene cloning)

vectors

DNA sequence used to carry other DNA


vectors

(gene cloning)

•can be put in a host easily•contains a replication origin•have a gene for screening

(eg. antibiotic resistance)


vectors

(gene cloning)

•for E. coli - plasmids bacteriophage M13


Fig. 10.5. Common cloning vectors for use with E. coli


vectors

(gene cloning)

put into cells via

transformationelectroporation


Fig. 10.7. Construction of recombinant DNA plasmids containing fragments derived from a donor organism


Fig. 10.4. Example of cloning


DNA to insert ?

(gene cloning)

libraries

genomiccDNA

collections of vectors (lots)each containing cloned DNA


genomic library (1)

(gene cloning)

phage

cut with restriction enzyme

x 10?

“sticky ends”


genomic library (2)

(gene cloning)

cut with same restriction enzyme

“sticky ends”


genomic library (3)

(gene cloning)

don’t forget DNA ligase

…lots of different vectors


cDNA library

(gene cloning)

eukaryotic DNA has lots of intronsgenes are very large

if we are only interested in the partof the gene that codes for protein…


cDNA library (1)

(gene cloning)

isolate the mRNA from the cell(s)

oligo-dT column


cDNA library (2)

(gene cloning)

5’-----------------AAAAAA-3’ mRNA use reverse transcriptase

3’-----------------TTTTTTT-5’ DNA5’ -----------------AAAAAA-3’ DNA

then DNA polymerase… …a double stranded DNA from each mRNA

complementary DNA - cDNA


cDNA library (3)

(gene cloning)

ligate DNAs into vectors


Fig. 10.8. Reverse transcriptase produces a single-stranded DNA complementary in sequence to a template RNA

10.1 Recombinant DNA Techniques(gene cloning)

transformationor

electroporation

mix vectors (with insert) with cells

libraries

collections of vectors withdifferent DNA inserts

genomiccDNA

great for abundant mRNA’s

libraries

mRNA in low copy number?

RT-PCR

reverse transcriptase-PCR

What do you need to know to do PCR?

More about plasmids

nice to have lots of different single-site RE sites

have to cut them open to put in insert

(directional cloning)


Fig. 10.9. (A) Diagram of the cloning vector pBluescript II (B) Sequence of the multiple cloning site showing the unique restriction sites [Data courtesy of Stratagene Cloning Systems, La Jolla, CA]

AATTC-our - DNA-A G-our - DNA-TTCGA

More about plasmids

(directional cloning)

…G…CTTAA

AATTCGATATCA GCTATAGTTCGA

AGCTT…A…

EcoRI HindIII

AATTC-our - DNA-A G-our - DNA-TTCGA

More about plasmids

need to screen for bacteriathat with the plasmid

need to have lots of different single site RE sites

you only want to grow the bacteria took up the plasmid


Fig. 10.9. (A) Diagram of the cloning vector pBluescript II (B) Sequence of the multiple cloning site showing the unique restriction sites [Data courtesy of Stratagene Cloning Systems, La Jolla, CA]

More about plasmids

need to screen for bacteriathat with the plasmid

need to screen for plasmids with an insert

need to have lots of different single site RE sites

some will have closed up without insert


Fig. 10.10A,B. Detection of recombinant plasmids through insertional inactivation of a fragment of the lacZ gene from E. coli


Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]

grow on ampicillin with Xgal



plasmid only

plasmid withinsert


Screening the library

106 to 10? of different clones

How do you “find” the one you want ?


Fig. 10.11. Colony hybridization


Chapter 10

Recombinant DNA Techniques10.1 cloning DNA- basics10.4 transgenic organisms - reverse genetics10.5 genetic engineering


10.4 Reverse genetics

In the past…

find mutant phenotype

find mutant gene

study wild-type gene



but now we can…

mutate a gene

find study the phenotype



Drosophila P elementsC. elegansmouse ESCdomestic animals

transforming the germ line



transposase

enzyme that can insert DNAflanked by inverted repeats

can place itself randomly into the chromosome


Fig. 10.18. Transformation in Drosophila mediated by the transposable element P

•remove some of the inverted repeats-cannot be inserted

and•insert DNA into coding region


Fig. 10.18. Transformation in Drosophila mediated by the transposable element P

your DNA + marker (eye color)



mouse

put DNA into fertilized eggusing engineered retrovirus

Embryonic stem cellsinsert modified cells into blastocyst


Fig. 10.19. Transformation of the germ line in the mouse using embryonic stem cells. [After M.R. Capecchi. 1989. Trends Genet. 5: 70.]



gene targeting

fig. 10.20


Fig. 10.20. Gene targeting in embryonic stem cells. [After M.R. Capecchi. 1989. Trends Genet. 5: 70.]



Ti plasmid used on plantsAgrobactgerium

fig. 10.21


Fig. 10.21. Transformation of a plant genome by T DNA from the Ti plasmid



Transformational rescue

fig. 10.22

by using inserts of different lengthsyou can find out how much of the DNA is necessary


Fig. 10.22. Genetic organization of the Drosophila gene white


Fig. 10.23. Eyes of a wildtype red-eyed male D. melanogaster and a mutant white-eyed male. [Courtesy of E. Lozovsky]


10.5 Genetic engineering applied

Animal growth rate

metallothionen promoter(very active)

growth hormone

Even though these Atlantic salmon are roughly the same age, the big one was genetically engineered to grow at twice the rate of normal salmon.

http://www.nytimes.com/2007/07/30/washington/30animal.html?_r=1&oref=slogin


plants

increase nutritional value

-caroteneprecursor to vitamin Ain yellow vegetables

high rice diets of lack -carotene

Fig. 10.25 Rice engineered to produce -carotene



rice with:b-carotene

plants



plants

rice also contains phytate which can causes iron deficiency

put in fungal gene to break down phytate and a gene to store iron and to promote iron absorption



rice rich in:b-caroteneiron

plants

added 6 genes from unrelated species



protein production

if we know the DNA sequence we transform cells to make the protein

human growth hormone,blood-clotting factors,insulin,…


Fig. 10.26. Relative numbers of patents issued for various clinical applications of the products of GE human genes. [Data from S. M. Thomas, et al., 1996. Nature 380: 387]



gene therapy

retroviruses

remove “bad” viral genesput in “fixed” sequencevirus will infect cell

and insert its’ new RNA



gene therapy

SCID

severe combined immuno- deficiency syndrome

(non-functional immune system)



gene therapy

SCID

gene(s) identified - ADAremove bone marrow cellsinfect with retrovirus having

fixed genereinsert cells

4/10 developed leukemia



vaccine production

production of “natural” vaccines is often dangerous

The end

Chapter 6

6.6 - 6.8 Practical applications of ourknowledge of DNA

structureGroup worksheet


Fig. 6.29. Structures of normal deoxyribose and the dideoxyribose sugar used in DNA sequencing


Fig. 6.30. Dideoxy method of DNA sequencing.

G A T C

(primer) 20 +

© 2006 Jones and Bartlett Publishers© 2006 Jones and Bartlett Publishers

Fig. 6.31. Florescence pattern trace obtained from a DNA sequencing gel

© 2006 jones and bartlett publishers chapter 10 recombinant dna techniques 10.1cloning dna- basics...

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