Recombinant DNA

Chapter 18

Learning Objectives

• Define Clone and DNA Cloning• List the three steps of production of recombinant

DNA• Describe the characteristics and uses of a

restriction endonuclease• Diagram the process of identifying a transformed

bacterial colony containing a gene of interest using Ampicillin Resistance, Lactose Metabolism plasmids and nucleic acid hybridization probes

Learning Objectives

• Explain the uses of RFLPs

• Describe the process of producing a transgenic organism, and explain its usefulness

DNA cloning

• Clone: genetically identical cells or individuals derived from a single ancestor

• DNA cloning: a method of producing a large amount of the DNA of interest

• Large amounts of identical pieces of DNA enable us to manipulate and recombine genetic material

DNA Technologies

• DNA technologies are used in molecular testing for many human genetic diseases

• DNA fingerprinting used to identify human individuals and individuals of other species

• Genetic engineering uses DNA technologies to alter the genes of a cell or organism

• DNA technologies and genetic engineering are a subject of public concern

Recombinant DNA

• DNA from two or more sources joined together

• DNA of interest can be spliced into bacterial plasmids (recombination)

• Plasmids replicate (amplification)

• Plasmids (DNA) are extracted (isolation)

Endonucleases

• Restriction enzymes (endunucleases) cut DNA at specific sequences in restriction sites– Restriction fragments result – Sticky ends have unpaired bases at cuts

which will hydrogen bond– Ligase stitches together paired sticky ends

Fig. 18-3, p. 374

Restriction sitefor EcoRI

DNA

Sticky end

Another DNA fragmentproduced by EcoRI digestion

Sticky end

Nick in sugar–phosphate backbone

Recombinant DNA molecule

EcoRI restriction enzyme cleaves sugar–phosphatebackbones at arrows.

DNA fragments with the same sticky ends can pair. Shown here is a DNA fragment inserting between two other DNA fragments, as happens when inserting a DNA fragment into a bacterial plasmid.

Nicks in sugar–phosphate backbonesare sealed by DNA ligase.

1

2

3

Recombinant DNA

• Restriction endonucleases

• Each type is specific for a four to eight base pair long palindromic recognition sequence of DNA

• Palindrome- reads the same on each strand 3’ to 5’ like GAATTC

CTTAAG

Fig. 18-4a, p. 375

Gene ofinterest

DNA fragments with sticky ends

Cell

Restrictionsite

Cut plasmid cloning vectors with a restriction

enzyme to produce sticky ends

Plasmidcloningvector

ampR

gene

lacZ+

gene

Fig. 18-2b, p. 373

5

Introduce recombinant moleculesinto bacterial cells; each bacteriumreceives a different plasmid. As thebacteria grow and divide, therecombinant plasmids replicate,thereby amplifying the piece of DNAinserted into the plasmid.

Identify the bacterium containingthe plasmid with the gene of interestinserted into it. Grow that bacteriumin culture to produce large amountsof the plasmid for experiments withthe gene of interest.

Inserted genomicDNA fragment

Bacterium

Bacterialchromosome

Progenybacteria

RecombinantDNA molecules

4

Recombinant DNA

• Break cells and use restriction enzyme to isolate DNA of interest (prokaryotic or eukaryotic)

• Insert into plasmid (recombination)• Transform into bacteria (replication)• Not very efficient, so for the third step (isolation)-

you need to have engineered a way to find the bacteria of interest

Four possibilities

1. Desired outcome: plasmid, lac+ broken, gene of interest inserted

2. Bacteria transformed with plasmid, but wrong gene inserted

3. Bacteria transformed with plasmid only- no gene at all inserted

4. Bacteria not transformed

Fig. 18-4b, p. 375

Resealed plasmid cloning vector with no inserted DNA fragment

Inserted DNA fragments with gene of interest

Recombinant plasmids

Inserted DNA fragment without gene of interest

Nonrecombinant plasmid

Recombinant DNA

• Insert into special screening plasmid- which contains the same restriction enzyme site used above, located in a lacZ gene.

• For recombination screening the lacZ gene is broken successfully, it will be white. If not, it will be blue.

• The plasmid also contain ampicillin resistance• If transformation worked, the bacteria will grow

on plates containing ampicillin. Those who were not transformed will not grow.

Fig. 18-4c, p. 375

Bacteria not transformedwith a plasmid

Bacteria transformed with plasmidsSelection:Transformed bacteria grow on medium containing ampicillin because of ampR gene on plasmid.

Screening:Blue colony contains bacteria with a non-recombinant plasmid; that is, the lacZ+

gene is intact.

Plate containingampicillin and X-gal

Untransformed bacterium

cannot grow on mediumcontaining ampicillin.

White colony contains bacteria with a recombinant plasmid; that is, the vector with an inserted DNA fragment. Once the white colony with the geneof interest is identified, it can be grown in culture to produce large quantities of the plasmid.

DNA Hybridization

• Uses nucleic acid probe to identify gene of interest in set of clones– Probe has tag for detection– Identified colony produces large quantities of

cloned gene

Fig. 18-5a, p. 377

Replica of bacterialcolonies

Culture mediumcontaining ampicillin

Filter paper

Filter paper

Bacterialcolony

Fig. 18-5b, p. 377

Labeled probe(single stranded)

Bag

Filter

Labeled single-stranded DNAprobe for thegene of interest

Hybridization has occurred between the labeled probe and the plasmids released from the bacteria in this colony. The hybridization is detected in subsequent steps.

Plasmid DNA(single stranded)

Fig. 18-5c, p. 377

Original master plate

Developedphotographicfilm

Corresponds toone colony onmaster plate

4 Possibilities

Outcome AMP LAC PROBE

Right Gene yes No Yes

Wrong Gene Yes No No

Plasmid only Yes Yes n/a

No Plasmid No No n/a

How else do we use

Restriction Endonuclease?

RFLPs

• Restriction fragment length polymorphisms– DNA sequence length changes due to varying

restriction sites from same region of genome– Sickle cell anemia has RFLPs

• Southern blot analysis uses electrophoresis, blot transfer, and labeled probes to identify RFLPs– Alternative is PCR and electrophoresis

Fig. 18-8, p. 381

β-Globin gene

175 bp

Normalallele

Sickle-cellmutant allele

Region of probeused to screen forsickle-cell mutation

201 bp

376 bp

MstII MstII

MstII MstII MstII

DNA Fingerprinting

• Distinguishes between individuals– Uses PCR at multiple loci within genome– Each locus heterozygous or homzygous for

short tandem repeats (STR)

• PCR amplifies DNA from STR– Number of gel electrophoresis bands shows

amplified STR alleles– 13 loci commonly used in human DNA

fingerprinting

Forensics and Ancestry

• Forensics compares DNA fingerprint from sample to suspect or victim– Usually reported as probability DNA came

from random individual

• Common alleles between children and parents used in paternity tests– Same principle used to determine

evolutionary relationships between species

Fig. 18-10a, p. 383

3 differentalleles

DNA

a. Alleles at an STR locus

Left PCR primer

STR locus

9 repeats Right PCR primer

11 repeats

15 repeats

Fig. 18-10b, p. 383

b. DNA fingerprint analysis of the STR locus by PCR

Cells ofthreeindividuals

Extract genomic DNAand use specificprimers to amplifythe STR locus usingthe PCR.

CBA

CBA

15,9 11,911,11

Anyalyze PCR product by gel electrophoresis

Positions corresponding to

alleles of STR locus

1511

9

Genetic Engineering

• Transgenic organisms – Modified to contain genes from external source

• Expression vector has promoter in plasmid for production of transgenic proteins in E. coli– Example: Insulin– Protocols to reduce risk of escape

Animal Genetic Engineering

• Transgenic animals used in research, correcting genetic disorders, and protein production

• Germ-line cell transgenes can be passed to offspring (somatic can not)– Embryonic germ-line cells cultured in quantity, made

into sperm or eggs– Stem cells

Fig. 18-11a, p. 385

Pure population oftransgenic cells

Germ-line cells derivedfrom mouse embryo

Transgene

Cell withtransgene

Fig. 18-11b, p. 385

Mice have transgenic cells inbody regions including germ line

Genetically engineeredoffspring—all cells transgenic

Gene Therapy

Attempts to correct genetic disorders– Germ-line gene therapy can’t be used on

humans– Somatic gene therapy used in humans

• Mixed results in humans– Successes for adenosine deaminase

deficiency (bubble kid) and sickle-cell– Deaths from immune response and leukemia-

like conditions– http://history.nih.gov/exhibits/genetics/sect4.htm

Animal Genetic Engineering

• “Pharm” animals produce proteins for humans – Usually produced in milk for harmless extraction

• Cloned mammals produced by implantation of diploid cell fused with denucleated egg cell– Low cloning success rate– Increased health defects in clones– Gene expression regulation abnormal

Learning Objectives

• Define Clone and DNA Cloning• List the three steps of production of recombinant

DNA• Describe the characteristics and uses of a

restriction endonuclease• Diagram the process of identifying a transformed

bacterial colony containing a gene of interest using Ampicillin Resistance, Lactose Metabolism plasmids and nucleic acid hybridization probes

Learning Objectives

• Explain the uses of RFLPs

• Describe the process of producing a transgenic organism, and explain its usefulness