Genetic Engineering
• Direct, deliberate modification of an organism’s genome
– bioengineering
• Biotechnology – use of an organism’s biochemical and metabolic pathways for industrial production
• Direct, deliberate modification of an organism’s genome
– bioengineering
• Biotechnology – use of an organism’s biochemical and metabolic pathways for industrial production
Practical Properties of DNA
• Intrinsic properties of DNA hold true even in a test tube.
• DNA heated from 90°C to 95°C; the two strands separate. The nucleotides can be identified, replicated, or transcribed.
• Slowly cooling the DNA allows complementary nucleotides to hydrogen bond and the DNA will regain double-stranded form
Enzymes for Dicing, Splicing, and Reversing Nucleic Acids
Restriction endonucleases – recognize specific sequences of DNA and break phosphodiester bonds between adjacent nucleotides
• The enzymes can be used to cleave DNA at desired sites.
• Recognize and clip the DNA at palindrome base sequences.
• Used in the lab to cut DNA into smaller pieces – restriction fragments
• Restriction fragments of varying lengths are called restriction fragment length polymorphisms (RFLPs).
Ligase – rejoins phosphate-sugar bonds
(sticky ends) cut by endonucleases
• Used for final splicing of genes into plasmids and chromosomes
Reverse transcriptase – makes a DNA copy of RNA – cDNA
• cDNA can be made from mRNA, tRNA, or rRNA
• Provides a means of synthesizing eucaryotic genes from mRNA transcripts – synthesized gene is free of introns
Methods for Analysis of DNA
• Gel electrophoresis - separates DNA fragments based on size
– DNA samples are placed on soft agar gel and subjected to an electric current.
– Negative charge of molecule causes DNA to move toward positive pole.
– Rate of movement is dependent on size of fragment – larger fragments move more slowly.
– Fragments are stained for observation.
– Useful in characterizing DNA fragments and comparing for genetic similarities
• Nucleic acid hybridization and probes
• Single-stranded DNA can unite with other single-stranded DNA, or RNA can unite with other RNA – hybridization
• Foundation for gene probes – short fragments of DNA of a known sequence that will base-pair with a stretch of DNA with a complementary sequence, if one exists in the sample
• Useful in detecting specific nucleotide sequences in unknown samples
– Southern blot method – DNA fragments are separated by electrophoresis, denatured and then incubated with DNA probes. Probes will attach to a complementary segment if present.
– isolate fragments from a mix of fragments and find specific gene sequences
• Hybridization test – used for diagnosing cause of infection and identifying unknown bacterium or virus
– DNA from test sample is isolated, denatured, placed on filter, and combined with microbe-specific probe
– commercially available diagnostic kits
Methods Used to Size, Synthesize, and Sequence DNA
DNA sequencing – determining the actual order and type of bases for all types of DNA
• Most common sequencing technique is Sanger technique
– Test strands are denatured to serve as a template to synthesize complementary strands.
– Fragments are divided into tubes that contain primers, DNA polymerase, all 4 nucleotides, and fluorescent labeled dideoxynucleotide.
Polymerase Chain Reaction (PCR) – method to amplify DNA; rapidly increases the amount of DNA in a sample
– Primers of known sequence are added, to indicate where amplification will begin, along with special heat tolerant DNA polymerase and nucleotides.
– repetitively cycled through denaturation, priming, and extension
– Each subsequent cycle doubles the number of copies for analysis.
– essentially important in gene mapping, the study of genetic defects and cancer, forensics, taxonomy, and evolutionary studies
Methods in Recombinant DNA Technology
• Recombinant DNA technology – the intentional removal of genetic material from one organism and combining it with that of a different organism
– Objective of recombinant technology is cloning which requires that the desired donor gene be selected, excised by restriction endonucleases, and isolated.
– The gene is inserted into a vector (plasmid, virus, cosmids) that will insert the DNA into a cloning host.
– Cloning host is usually bacterium or yeast that can replicate the gene and translate it into a protein product.
Characteristics of Cloning Vectors
• Must be capable of carrying a significant piece of donor DNA
• Must be readily accepted by the cloning host
• Plasmids – small, well characterized, easy to manipulate and can be transferred into appropriate host cells through transformation
• Bacteriophages – have the natural ability to inject their DNA into bacterial hosts through transduction
Vector Considerations
• Origin of replication is needed so it will be replicated.
• Vector must accept DNA of the desired size.
• Gene which confers drug resistance to their cloning host
Characteristics of Cloning Hosts
Construction of a Recombinant, Insertion, and Genetic Expression
• Prepare the isolated genes for splicing into a vector by digesting the gene and the plasmid with the same restriction endonuclease enzymes creating complementary sticky ends on both the vector and insert DNA.
• The gene and plasmid are placed together, their free ends base-pair, and ligase joins them.
• The gene and plasmid combination is a recombination.
• The recombinant is introduced into a cloning host.
Biochemical Products of Recombinant DNA Technology
• Enables large scale manufacturing of life-saving hormones, enzymes, vaccines
– insulin for diabetes
– human growth hormone for dwarfism
– erythropoietin for anemia
– Factor VIII for hemophilia
– HBV vaccine
Genetically Modified Organisms (GMO)
• Recombinant microbes
– Pseudomonas syringae – prevents ice crystals
– Bacillus thuringienisis –encodes an insecticide
• Transgenic plants
– rice that makes beta-carotene
– tobacco resistant to herbicides
– peas resistant to weevils
• Transgenic animals
– mouse models for CF, Alzheimer’s, sickle cell anemia
– sheep or goats that make medicine in their milk semen
Genome Analysis
• DNA Fingerprinting –
– Every individual has a unique sequence of DNA.
– used to:
• identify hereditary relationships
• study inheritance of patterns of diseases
• study human evolution
• identify criminals or victims of disaster
• Analysis of mitochondrial DNA is used to trace evolutionary origins.
• microarray analysis – track the expression of genes; used to identify and devise treatments for diseases based on the genetic profile of the disease
• Intrinsic properties of DNA hold true even in a test tube.
• DNA heated from 90°C to 95°C; the two strands separate. The nucleotides can be identified, replicated, or transcribed.
• Slowly cooling the DNA allows complementary nucleotides to hydrogen bond and the DNA will regain double-stranded form
Enzymes for Dicing, Splicing, and Reversing Nucleic Acids
Restriction endonucleases – recognize specific sequences of DNA and break phosphodiester bonds between adjacent nucleotides
• The enzymes can be used to cleave DNA at desired sites.
• Recognize and clip the DNA at palindrome base sequences.
• Used in the lab to cut DNA into smaller pieces – restriction fragments
• Restriction fragments of varying lengths are called restriction fragment length polymorphisms (RFLPs).
Ligase – rejoins phosphate-sugar bonds
(sticky ends) cut by endonucleases
• Used for final splicing of genes into plasmids and chromosomes
Reverse transcriptase – makes a DNA copy of RNA – cDNA
• cDNA can be made from mRNA, tRNA, or rRNA
• Provides a means of synthesizing eucaryotic genes from mRNA transcripts – synthesized gene is free of introns
Methods for Analysis of DNA
• Gel electrophoresis - separates DNA fragments based on size
– DNA samples are placed on soft agar gel and subjected to an electric current.
– Negative charge of molecule causes DNA to move toward positive pole.
– Rate of movement is dependent on size of fragment – larger fragments move more slowly.
– Fragments are stained for observation.
– Useful in characterizing DNA fragments and comparing for genetic similarities
• Nucleic acid hybridization and probes
• Single-stranded DNA can unite with other single-stranded DNA, or RNA can unite with other RNA – hybridization
• Foundation for gene probes – short fragments of DNA of a known sequence that will base-pair with a stretch of DNA with a complementary sequence, if one exists in the sample
• Useful in detecting specific nucleotide sequences in unknown samples
– Southern blot method – DNA fragments are separated by electrophoresis, denatured and then incubated with DNA probes. Probes will attach to a complementary segment if present.
– isolate fragments from a mix of fragments and find specific gene sequences
• Hybridization test – used for diagnosing cause of infection and identifying unknown bacterium or virus
– DNA from test sample is isolated, denatured, placed on filter, and combined with microbe-specific probe
– commercially available diagnostic kits
Methods Used to Size, Synthesize, and Sequence DNA
DNA sequencing – determining the actual order and type of bases for all types of DNA
• Most common sequencing technique is Sanger technique
– Test strands are denatured to serve as a template to synthesize complementary strands.
– Fragments are divided into tubes that contain primers, DNA polymerase, all 4 nucleotides, and fluorescent labeled dideoxynucleotide.
Polymerase Chain Reaction (PCR) – method to amplify DNA; rapidly increases the amount of DNA in a sample
– Primers of known sequence are added, to indicate where amplification will begin, along with special heat tolerant DNA polymerase and nucleotides.
– repetitively cycled through denaturation, priming, and extension
– Each subsequent cycle doubles the number of copies for analysis.
– essentially important in gene mapping, the study of genetic defects and cancer, forensics, taxonomy, and evolutionary studies
Methods in Recombinant DNA Technology
• Recombinant DNA technology – the intentional removal of genetic material from one organism and combining it with that of a different organism
– Objective of recombinant technology is cloning which requires that the desired donor gene be selected, excised by restriction endonucleases, and isolated.
– The gene is inserted into a vector (plasmid, virus, cosmids) that will insert the DNA into a cloning host.
– Cloning host is usually bacterium or yeast that can replicate the gene and translate it into a protein product.
Characteristics of Cloning Vectors
• Must be capable of carrying a significant piece of donor DNA
• Must be readily accepted by the cloning host
• Plasmids – small, well characterized, easy to manipulate and can be transferred into appropriate host cells through transformation
• Bacteriophages – have the natural ability to inject their DNA into bacterial hosts through transduction
Vector Considerations
• Origin of replication is needed so it will be replicated.
• Vector must accept DNA of the desired size.
• Gene which confers drug resistance to their cloning host
Characteristics of Cloning Hosts
- Rapid overturn, fast growth rate
- Can be grown in large quantities using ordinary culture methods
- Nonpathogenic
- Genome that is well delineated
- Capable of accepting plasmid or bacteriophage vectors
- Maintains foreign genes through multiple generations
Construction of a Recombinant, Insertion, and Genetic Expression
• Prepare the isolated genes for splicing into a vector by digesting the gene and the plasmid with the same restriction endonuclease enzymes creating complementary sticky ends on both the vector and insert DNA.
• The gene and plasmid are placed together, their free ends base-pair, and ligase joins them.
• The gene and plasmid combination is a recombination.
• The recombinant is introduced into a cloning host.
Biochemical Products of Recombinant DNA Technology
• Enables large scale manufacturing of life-saving hormones, enzymes, vaccines
– insulin for diabetes
– human growth hormone for dwarfism
– erythropoietin for anemia
– Factor VIII for hemophilia
– HBV vaccine
Genetically Modified Organisms (GMO)
• Recombinant microbes
– Pseudomonas syringae – prevents ice crystals
– Bacillus thuringienisis –encodes an insecticide
• Transgenic plants
– rice that makes beta-carotene
– tobacco resistant to herbicides
– peas resistant to weevils
• Transgenic animals
– mouse models for CF, Alzheimer’s, sickle cell anemia
– sheep or goats that make medicine in their milk semen
Genome Analysis
• DNA Fingerprinting –
– Every individual has a unique sequence of DNA.
– used to:
• identify hereditary relationships
• study inheritance of patterns of diseases
• study human evolution
• identify criminals or victims of disaster
• Analysis of mitochondrial DNA is used to trace evolutionary origins.
• microarray analysis – track the expression of genes; used to identify and devise treatments for diseases based on the genetic profile of the disease