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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 - PowerPoint PPT Presentation
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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