L3 Recombinant DNA Cloning HT12 Bilda

Transfer of genetic information by DNA

2008 Sinauer Associates, Inc. F. Griffith, 1928, J. of Hygiene


2008 Sinauer Associates, Inc.

Avery O.T. et al, 1944, J. of Experimental Medicine

Recombinant DNA

Recombinant DNA technology provided scientists with the ability to isolate, sequence, and manipulate individual genes from any type of cell. !

It has enabled detailed molecular studies of the structure and function of eukaryotic genes and genomes, and revolutionized our understanding of cell biology.!

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Generation of a recombinant DNA molecule

A molecular tool box

Enzymes -restriction endonuclease -ligase -DNA polymerase -reverse transcriptase (RT) -kinase -polynucleotide transferase -phosphatase

Oligonucleotide synthesis -primers -probes -gene assembly

DNA sequencing -Maxam-Gilbert -Sanger -pyrosequencing

PCR In vitro mutagenesis Vectors -plasmids -phages (M13, ) -cosmides -YACs -viruses

Host cells -bacteria -yeast/fungus/molds -plant cells -insect cells -mammalian cells

Transformation -heat-shock -electroporation -gene gun -micro injection -transfecion

Selection methods -antibiotic resistance -genetic complementation

Screening methods -genotypic -phenotypic

Enzymes

Kloning - Expression

Restriction enzymes Specific DNA sequences (4-8 bp) are recognized and cleaved by restriction endonucleases (RE) Recognition sequence (RS) is often palindromic (can be read from both ends) Is part of the defense system against foreign DNA; > hundreds of different RE Specific methylases methylates certain bases in the RS, whereby the bacteriums own DNA becomes protected from cleavage

EcoRV EcoRI PstI

GAATTC CTTAAG GATATC CTATAG CTGCAG GACGTC

GAT ATC TAG G CTTAA AATTC G CTGCA G ACGTC G

blunt end sticky ends CTA

Recognition sites of common RE

Restriction map EcoRI recognizes the sequence GAATTC. !This sequence is present at five sites in DNA of the bacteriophage , so EcoRI digests DNA into six

fragments ranging from 3.6 to 21.2 kilobases long.!

Restriction maps of and adenovirus DNAs

For larger DNA molecules such as cellular genomes, restriction endonuclease digestion alone does not provide sufficient resolution.!

For example, the human genome would yield more than 500,000 EcoRI fragments.!

Restriction enzymes

Ligase Ligases recreate phosphodiesterbonds by joining the 5-phosphate group to the free 3-hydroxyl group. A common enzyme is the T4-DNA ligase. Ligases are used to glue DNA fragments to each other.

G-OH CCTAG-P P-GATCC HO-G + T4-DNA ligase, ATP

G CCTAG GATCC G

G-OH CCTAG-P P-GATCT HO-A + T4-DNA ligase, ATP

G CCTAG GATCT A can be re-cleaved by BamHI

(5-GGATCC-3) cannot be re-cleaved by BamHI

(5-GGATCC-3)

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Ligase reaction

DNA polymerases DNA polymerases incorporates deoxynucleotides (dNTPs) to a growing DNA chain; the dNTPs are added to the free 3-OH group using the opposite strand as template. Some frequently used polymerases are DNA polymerase I, T7-DNA polymerase, and Taq-polymerase.

They are used for several things, for example: - generation of blunt-ends - in DNA sequencing reactions

G CCATG + dNTPs, polymerase GGTAC CCATG

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Reverse transcription and retrovirus replication

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Reverse transcriptase (RT) Reverse transcriptase (RT) synthesizes a complementary DNA chain from a RNA template. Retroviruses (e.g., HIV) use this mechanism to make a DNA copy of its RNA genome.

Can be used to our advantage: - in cDNA synthesis

3 5 Cap AAAAA mRNA

3 5 Cap AAAAA

TTTTT 5

TTTTT 5 3 cDNA mRNA

reverse transcriptase + dNTPs

Terminal transferase Terminal transferase adds nucleotides to the 3-end of DNA chains.

Used, among other things, for generation of cohesive ends from blunt-end fragments and also at one step in cDNA synthesis

5 3

3

5

5 3

5 3

3

5

3

5

+

terminal transferase

dATP dTTP

AAAA-OH HO-TTTT

AAAA

TTTT

ligation

Phosphatases och kinases

Phosphatases remove free 5-phosphate groups from DNA - used to minimize the incidence of self-ligated vector in DNA cloning - commonly used phosphatases are the shrimp- and bovine alkaline phosphatase

Kinases adds phosphate groups to the 5-end of DNA - used to phosphorylate synthetic DNA (e.g., linkers) before ligation - polynucleotidekinase (PNK)

Vectors


Cloning vectors: desired properties

small universal; should work in different organisms easy to isolate from the host organism easy to detect and select multiple copies (is usually advantageous) several unique RE localized to a specific region (mcs) convenient method for detection of cloned DNA

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Plasmids Plasmids are circular extrachromosomal DNA Used as vectors = carriers of foreign DNA - used in cloning and recombinant protein expression - insert size 5 kb

Plasmids usually contain: - ori (origin of replication) - gene/s for selection (often antibiotic resistance genes; bla, cat) - multiple cloning site (mcs) - sometimes a gene that allows for screening (e.g., lacZ for blue-white screening)

Different plasmid-types have different copy nr Plasmids belong to different incompatibility groups; two plasmids belonging to the same group cannot be stably propagated simultaneously within a single cell.

Figure 8-40 Molecular Biology of the Cell ( Garland Science 2008)

Plasmid preparation

Bacteriophage

Figure 5-78 Molecular Biology of the Cell ( Garland Science 2008)

Bacteriophage -life cycle

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Bacteriophage Bacteriophage is a virus that infects bacteria (E. coli). - a lytic and a lysogenic phase (prophage) in the life cycle. - genome size approx. 45 kb; a central region of 15 kb is not essential for replication. - have complementary single stranded cohesive ends (COS-sites); used by the phage to make concatamers of its genome when the DNA is packed into phage particles.

the genome has been modified to work better as a vector; unique sites that flank the central region have been introduced to simplify DNA cloning/replacement. - inserts must have a certain size (approx. 15 kb), or else no infectious phage particles can be formed.

used for cloning of genomic fragments, since plasmids are not suitable for cloning of larger DNA fragments.

Biotechnology: Applying the genetic revolution, Figure 3.17

In vitro packaging:bacteriophage

Cosmids Cosmids are plasmid-phage hybrids. They contain: - ori - gene for antibiotic resistance (e.g., Tetr) - cos-sites - cloning sites

Cosmids are used for cloning of genomic DNA fragments larger than 15 kb (this is the limit of what phage can harbor). Cosmids can accommodate an insert size of approx. 45 kb.

vector DNA (cosmid) cos

genomic DNA antibiotic resistance

gene

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Yeast artificial chromosome (YAC) YACs contain: - autonomously replicating sequence (ARS) from yeast chromosome - centromer (CEN); ensures stable and even distribution of the YACs between mother and daughter cells - two telomers; constitute chromosome ends and enables replication of the YACs as small linear chromosomes. - selectable gene markers, e.g., LEU2 that complement Leu negative strains. - cloning sites

YACs used for cloning of very large genomic fragments (100-2000 kb) telomer LEU2 ARS CEN jst DNA

50 kb

telomer

Vectors for cloning large DNA fragments

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Various cloning vectors


Host cells


Different vectors are used to introduce recombinant DNA in various types of host cells, for example:!

! !! Baceria !

Yeast! Insect cells! Mammalian cells! Plant cells!

Eukaryotic cells! Post-translational modifications!(e.g., glycosylations) possible !

Host cells

- E. coli the most common!- High expression levels !- Relatively easy to scale-up the production process. !

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Transformation methods


Heat shock transformation


Electroporation

Issuses to consider:!1. Cell size!2. Temperature!3. Post-pulse manipulation!4. Composition of electrodes and pulsing medium!

Acta Physiol Scand. 2003 Apr;177(4):437-47

Selection methods


Antibiotic resistance !- ampicillin!- carbenicillin!- chloramphenicol!- tetracyclin!- kanamycin!

Inhibits cell wall synthesis!

Inhibits protein synthesis!

Gene for !antibiotic-!resistance!

Cloned gene!

Plasmid!

Transform bacteria with ligated plasmid!

Spread bacteria and grow on solid growth media (agar) supplemented with antibiotics!

Only bacteria containing the plasmid with the antibiotic resistance gene survive and form colonies!

Commonly used antibiotics in molecular biology: !

Screening methods


Genotypic screening

Probe hybridization! known nucleotide sequence (gene sequence)!

15-18 nt long probe! unknown nucleotide sequence (gene sequence)!

guessmerprobe, approx. 50 nt long (unique)"!

degenerate probe, approx. 18 nt long"based on amino acid sequence (6 residues long)!

Screening a recombinant library by hybridization

Phenotypic screening

Activity based! e.g., protease activity (subtilisin)!

grow on casein media and a clear zone (halo) will appear around proteolytically active clones!

Immuno based! antibody specific for the protein of interest!

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EcoRI site!

LacZ!AmpR!

Cloning of a DNA fragment in the EcoRI site in LacZ (encodes the enzyme -galactosidase)!

Transform bacteria and grow on agar plates supplemented w ampicillin and X-gal!

- only the bacteria that harbor the plasmid are resistant to ampicillin and can grow!!- bacteria w/o cloned fragment have a functional -galactosidase gene and convert the color-less substrate X-gal to a blue-colored product!!- in bacteria w a cloned fragment the -galaktosidase gene is destroyed and therefore only give rise to white colonies!

Blue-white screening

OOH

H

O

HH

OHH

OHH

OH

HN

ClBr

X-gal"

An example of gene cloning


Gene library

Creating a DNA Library: Genomic DNA from the chosen organism is first partially digested with a restriction enzyme that recognizes a four base-pair sequence. Partial digestions are preferred because some of the restriction enzyme sites are not cut, and larger fragments are generated. If every recognition site were cut by the restriction enzyme, then the genomic DNA would not contain many whole genes. The genomic fragments are cloned into an appropriate vector, and transformed and maintained in bacteria.

Gene library

Chromosome digested w RE (4-cutter) ! (4x4x4x4=256 bp) [e.g., Sau3AI, ^GATC^]!

Partial digestion! Large randomly digested gene fragments (~2500 bp)!

Plasmid cleaved w compatible RE (6-cutter)! Matching overlaps [e.g., BamHI, G^GATC^C]!

Ligation (Phosphatase treated plasmid)! Transformation (CaCl2, Electroporation)! Selection!

Colonies with recombinant plasmid!

Cloning of a prokaryotic gene Example: Subtilisin!

from Bacillus subtilis! protease! gene size, approx. 1000 bp! used in washing powders!

Sub tasks! create a gene library (genomic DNA)! selection and genotypic screening! phenotypic screening! sequence verification! clone into an expression vector! activity studies/protein engineering!

Cloning of a eukaryotic gene Often mRNA as source! Sub tasks!

Create a gene library! mRNA purification! cDNA synthesis (S1-method, RNaseH-method)! Ligation into a vector (phage or plasmid)!

Selection and genotypic screening! Phenotypic screening! Sequence verification! Expression vector (choice of host!)! Activity studies/protein engineering!

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L3 Recombinant DNA Cloning HT12 Bilda