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Recombinant DNA I Basics of molecular cloning Polymerase chain reaction cDNA clones and screening

Recombinant DNA I

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Recombinant DNA I. Basics of molecular cloning Polymerase chain reaction cDNA clones and screening. Recombinant DNA Technology. - PowerPoint PPT Presentation

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Page 1: Recombinant DNA I

Recombinant DNA I

Basics of molecular cloning

Polymerase chain reaction

cDNA clones and screening

Page 2: Recombinant DNA I

Recombinant DNA Technology

• Utilizes microbiological selection and screening procedures to isolate a gene that represents as little as 1 part in a million of the genetic material in an organism.

• DNA from the organism of interest is divided into small pieces that are then placed into individual cells (usually bacterial).

• These can then be separated as individual colonies on plates, and they can be screened to find the gene of interest.

• This process is also called molecular cloning.

Page 3: Recombinant DNA I

DNA pieces are joined in vitro to form recombinant molecules

• Generate sticky ends on the DNA, e.g. with restriction endonucleases

• Tie DNA molecules from different sources together with DNA ligase

Page 4: Recombinant DNA I

Restriction endonucleases generate ends that facilitate mixing and matching

GAATTCCTTAAG

GAATTCCTTAAG

GCTTAA

AATTC G

GCTTAA

AATTC G

EcoRI cut

Mix and ligate

GCTTAAAATTC G

GCTTAAAATTC G

Recombinant molecules

GAATTCCTTAAG

GAATTCCTTAAG

Parental molecules

Page 5: Recombinant DNA I

DNA ligase covalently joins two DNA molecules

• Uses ATP or NADH to provide energy to seal nicks

P P P

P

P P P P P P P

P P P P P P

P

PP P P

A G G A A T T C G T A

T C C T T A A G C A T

OH

OH

nick

nick

P P PP

P P P P P P P

P P P P P P P

P

P P P

A G G A A T T C G T A

T C C T T A A G C A T

T4 DNA ligase + ATP

Page 6: Recombinant DNA I

Alternate method to join DNA: homopolymer tails

Page 7: Recombinant DNA I

Alternate method to join DNA:

linkers

Page 8: Recombinant DNA I

Introduction of recombinant DNA into living cells via vectors

• Autonomously replicating DNA molecules– (have an origin of replication)

• Selectable marker, such as drug resistance

• Insertion site for foreign DNA– (often a genetically engineered multiple cloning

region with sites for several restriction enzymes)

Page 9: Recombinant DNA I

Plasmid vectors

• Circular, extrachromosomal, autonomously replicating DNA molecules

• Frequently carry drug resistance genes

• Can be present in MANY copies in the cell

Page 10: Recombinant DNA I

A common plasmid cloning vector: pUC

ColE1 originof replication

lacZ

mulitplecloning sites

ApR

pUC

ColE1 ori

lacZ

ApR

pUC recombinant

Lac+, or blue colonieson X-gal in appropriatestrains of E. coli

Lac-, or white colonieson X-gal in appropriatestrains of E. coli

foreign DNA

High copy number

Page 11: Recombinant DNA I

Transformation of E. coli

• E. coli does NOT have a natural system to take up DNA

• Treat with inorganic salts to destabilize cell wall and cell membrane

• During a brief heat shock, some of the bacteria takes up a plasmid molecule

• Can also use electroporation

Page 12: Recombinant DNA I

Phage vectors

• More efficient introduction of DNA into bacteria

• Lambda phage and P1 phage can carry large fragments of DNA– 20 kb for lambda– 70 to 300 kb for P1

• M13 phage vectors can be used to generate single-stranded DNA

Page 13: Recombinant DNA I

YAC vectors for cloning large DNA inserts

CEN4ori

URA3

TEL TEL

TRP1

CEN4ori

SUP4

TRP1

URA3TEL TEL

pYAC3

BB

S

Cut with restrictionEnzymes S + B

Ligate to very largeFragments of genomicDNA

Large insert, 400 to as much as 1400 kb

11.4 kb

Yeast artificial chromosome = YAC

Not to scale.

Page 14: Recombinant DNA I

Bacterial artificial chromosomes

• Are derived from the fertility factor, or F-factor, of E. coli

• Can carry large inserts of foreign DNA, up to 300 kb

• Are low-copy number plasmids

• Are less prone to insert instability than YACs

• Have fewer chimeric inserts (more than one DNA fragment) than YACs

• Extensively used in genome projects

Page 15: Recombinant DNA I

BAC vectors for large DNA inserts

Cut with restriction enzyme E, remove “stuffer”

Ligate to very large fragments of genomic DNA

SacBII

promoter

oriF

Cm(R)

pBACe3.6

EES

11.5 kb

SacB+: SacBII encodes levansucrase, which converts sucrose to levan, a compound toxic to the bacteria.

Large insert, 300kb

oriFCm(R)

promoter

SSacBII

SacB-: No toxic levan produced on sucrosemedia: positive selection for recombinants.

Not to scale.

Page 16: Recombinant DNA I

PCR provides access to specific DNA segments

• Polymerase Chain Reaction

• Requires knowledge of the DNA sequence in the region of interest.

• As more sequence information becomes available, the uses of PCR expand.

• With appropriate primers, one can amplify the desired region from even miniscule amounts of DNA.

• Not limited by the distribution of restriction endonuclease cleavage sites.

Page 17: Recombinant DNA I

Polymerase chain reaction, cycle 1Primer 1 Primer 2

Template

1. Denature

2. Anneal primers

3. Synthesize new DNA with polymerase

Cycle 1

Page 18: Recombinant DNA I

Polymerase chain reaction, cycle 21. Denature

2. Anneal primers

3. Synthesize new DNA with polymerase

Cycle 2

Page 19: Recombinant DNA I

PCR, cycle 3

1. Denature

2. Anneal primers

3. Synthesize new DNA with polymerase

Cycle 3 (focus on DNA segments bounded by primers)

2 duplex molecules of desired product

Page 20: Recombinant DNA I

PCR, cycle 4: exponential increase in product

Cycle 4: Denature, anneal primers, and synthesize new DNA:

6 duplex molecules of desired product

Page 21: Recombinant DNA I

PCR, cycle 5: exponential increase in product

Cycle 5: Denature, anneal primers, and synthesize new DNA:

14 duplex molecules of desired product

Page 22: Recombinant DNA I

PCR: make large amounts of a particular sequence

• The number of molecules of the DNA fragment between the primers increases about 2-fold with each cycle.

• For n = number of cycles, the amplification is approximately [2exp(n-1)]-2.

• After 21 cycles, the fragment has been amplified about a million-fold.

• E.g. a sample with 0.1 pg of the target fragment can be amplified to 0.1 microgram

Page 23: Recombinant DNA I

PCR is one of the most widely used molecular tools in biology

• Molecular genetics - obtain a specific DNA fragment– Test for function, expression, structure, etc.

• Enzymology - place fragment encoding a particular region of a protein in an expression vector

• Population genetics - examine polymorphisms in a population

• Forensics - test whether suspect’s DNA matches DNA extracted from evidence at crime scene

• Etc, etc