11 9 09 lecture slides
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10.2 DNA and RNA are polymers of nucleotides
– The monomer unit of DNA and RNA is the nucleotide, containing
– Nitrogenous base– 5-carbon sugar– Phosphate group
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– DNA and RNA are polymers called polynucleotides
– A sugar-phosphate backbone is formed by covalent bonding between the phosphate of one nucleotide and the sugar of the next nucleotide
– Nitrogenous bases extend from the sugar-phosphate backbone
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Sugar-phosphate backbone
DNA nucleotide
Phosphate group
Nitrogenous baseSugar
DNA polynucleotide
DNA nucleotide
Sugar(deoxyribose)
Thymine (T)
Nitrogenous base(A, G, C, or T)
Phosphategroup
Sugar(deoxyribose)
Thymine (T)
Nitrogenous base(A, G, C, or T)
Phosphategroup
Pyrimidines
Guanine (G)Adenine (A)Cytosine (C)Thymine (T)
Purines
Sugar(ribose)
Uracil (U)
Nitrogenous base(A, G, C, or U)
Phosphategroup
Ribose
Cytosine
Uracil
Phosphate
Guanine
Adenine
Hydrogen bond
Basepair
Partial chemical structure Computer modelRibbon model
Basepair
Ribbon model
Hydrogen bond
Partial chemical structure
THE FLOW OF GENETIC INFORMATION FROM DNA
TO RNA TO PROTEIN
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10.6 The DNA genotype is expressed as proteins, which provide the molecular basis for phenotypic traits
– A gene is a sequence of DNA that directs the synthesis of a specific protein
– DNA is transcribed into RNA– RNA is translated into protein
– The presence and action of proteins determine the phenotype of an organism
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10.6 The DNA genotype is expressed as proteins, which provide the molecular basis for phenotypic traits
– Demonstrating the connections between genes and proteins
– The one gene–one enzyme hypothesis was based on studies of inherited metabolic diseases
– The one gene–one protein hypothesis expands the relationship to proteins other than enzymes
– The one gene–one polypeptide hypothesis recognizes that some proteins are composed of multiple polypeptides
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Cytoplasm
Nucleus
DNA
Cytoplasm
Nucleus
DNA
Transcription
RNA
Cytoplasm
Nucleus
DNA
Transcription
RNA
Translation
Protein
10.7 Genetic information written in codons is translated into amino acid sequences
– The sequence of nucleotides in DNA provides a code for constructing a protein
– Protein construction requires a conversion of a nucleotide sequence to an amino acid sequence
– Transcription rewrites the DNA code into RNA, using the same nucleotide “language”
– Each “word” is a codon, consisting of three nucleotides
– Translation involves switching from the nucleotide “language” to amino acid “language”
– Each amino acid is specified by a codon– 64 codons are possible
– Some amino acids have more than one possible codon
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Polypeptide
Translation
Transcription
DNA strand
Codon
Amino acid
RNA
Polypeptide
Translation
Transcription
Gene 1
DNA molecule
DNA strand
Codon
Amino acid
Gene 2
Gene 3
RNA
GENE CLONING
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12.1 Genes can be cloned in recombinant plasmids
– Genetic engineering involves manipulating genes for practical purposes
– Gene cloning leads to the production of multiple identical copies of a gene-carrying piece of DNA
– Recombinant DNA is formed by joining DNA sequences from two different sources
– One source contains the gene that will be cloned
– Another source is a gene carrier, called a vector
– Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors
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– Steps in cloning a gene1. Plasmid DNA is isolated
2. DNA containing the gene of interest is isolated
3. Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle
4. DNA with the target gene is treated with the same enzyme and many fragments are produced
5. Plasmid and target DNA are mixed and associate with each other
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12.1 Genes can be cloned in recombinant plasmids
6. Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together
7. The recombinant DNA is taken up by a bacterial cell
8. The bacterial cell reproduces to form a clone of cells
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12.1 Genes can be cloned in recombinant plasmids
Animation: Cloning a Gene
Examples ofgene use
RecombinantDNAplasmid
E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Geneof interest
Cell with DNAcontaining geneof interest
Recombinantbacterium
Cloneof cells
Genes may be insertedinto other organisms
Genes or proteinsare isolated from thecloned bacterium
Harvestedproteinsmay be used directly
Examples ofprotein use
Gene of interest
Isolateplasmid
1
IsolateDNA
2
Cut plasmidwith enzyme
3
Cut cell’s DNAwith same enzyme
4
Combine targeted fragmentand plasmid DNA
5
Add DNA ligase,which closesthe circle withcovalent bonds
6
Put plasmidinto bacteriumby transformation
7
Allow bacteriumto reproduce
8
9
E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Cell with DNAcontaining geneof interest
Isolateplasmid
IsolateDNA
1
2
E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Cell with DNAcontaining geneof interest
Gene of interest
Isolateplasmid
IsolateDNA
Cut plasmidwith enzyme
Cut cell’s DNAwith same enzyme
1
2
3
4
E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Cell with DNAcontaining geneof interest
Gene of interest
Isolateplasmid
IsolateDNA
Cut plasmidwith enzyme
Cut cell’s DNAwith same enzyme
1
2
3
4
Combine targeted fragmentand plasmid DNA
5
E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Cell with DNAcontaining geneof interest
Gene of interest
Isolateplasmid
IsolateDNA
Cut plasmidwith enzyme
Cut cell’s DNAwith same enzyme
1
2
3
4
RecombinantDNAplasmid
Geneof interest
Combine targeted fragmentand plasmid DNA
Add DNA ligase,which closesthe circle withcovalent bonds
5
6
RecombinantDNAplasmid
Geneof interest
Recombinantbacterium
Put plasmidinto bacteriumby transformation
7
RecombinantDNAplasmid
Geneof interest
Recombinantbacterium
Cloneof cells
Put plasmidinto bacteriumby transformation
Allow bacteriumto reproduce
8
7
RecombinantDNAplasmid
Geneof interest
Recombinantbacterium
Cloneof cells
Genes or proteinsare isolated from thecloned bacterium
Harvestedproteinsmay be used directly
Examples ofprotein use
Put plasmidinto bacteriumby transformation
Allow bacteriumto reproduce
8
7
Genes may be insertedinto other organisms
Examples ofgene use
9
12.8 CONNECTION: Genetically modified organisms are
transforming agriculture – Genetically modified (GM) organisms contain one or more genes introduced by artificial means
– Transgenic organisms contain at least one gene from another species
– GM plants– Resistance to herbicides– Resistance to pests– Improved nutritional profile
– GM animals– Improved qualities– Production of proteins or therapeutics
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Agrobacterium tumefaciens
DNA containinggene for desired trait
Tiplasmid Insertion of gene
into plasmid
RecombinantTi plasmid
1
Restriction site
Agrobacterium tumefaciens
DNA containinggene for desired trait
Tiplasmid Insertion of gene
into plasmid
RecombinantTi plasmid
1
Restriction site
Plant cell
Introductioninto plantcells
2
DNA carrying new gene
Agrobacterium tumefaciens
DNA containinggene for desired trait
Tiplasmid Insertion of gene
into plasmid
RecombinantTi plasmid
1
Restriction site
Plant cell
Introductioninto plantcells
2
DNA carrying new gene
Regenerationof plant
3
Plant with new trait
12.9 Genetically modified organisms raise concerns
about human and environmental health – Scientists use safety measures to guard against
production and release of new pathogens
– Concerns related to GM organisms– Can introduce allergens into the food supply
– FDA requires evidence of safety before approval
– Exporters must identify GM organisms in food shipments
– May spread genes to closely related organisms– Hybrids with native plants may be prevented by modifying GM
plants
– Regulatory agencies address the safe use of biotechnology
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– Advantages of PCR– Can amplify DNA from a small sample
– Results are obtained rapidly
– Reaction is highly sensitive, copying only the target sequence
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12.12 The PCR method is used to amplify DNA sequences
Cycle 1yields 2 molecules
21 3
GenomicDNA
Cycle 3yields 8 molecules
Cycle 2yields 4 molecules
3 5 3 5 3 5
Targetsequence
Heat toseparateDNA strands
Cool to allowprimers to formhydrogen bondswith ends oftarget sequences
35
3 5
35
35 35
Primer New DNA
5
DNApolymerase addsnucleotidesto the 3 endof each primer
5
Cycle 1yields 2 molecules
GenomicDNA
3 5 3 5 3 5
Targetsequence
Heat toseparateDNA strands
Cool to allowprimers to formhydrogen bondswith ends oftarget sequences
35
3 5
35
35 35
Primer New DNA
5
DNApolymerase addsnucleotidesto the 3 endof each primer
215
3
Cycle 3yields 8 molecules
Cycle 2yields 4 molecules
12.13 Gel electrophoresis sorts DNA molecules by size
– Gel electrophoresis separates DNA molecules based on size
– DNA sample is placed at one end of a porous gel– Current is applied and DNA molecules move from the
negative electrode toward the positive electrode– Shorter DNA fragments move through the gel pores
more quickly and travel farther through the gel – DNA fragments appear as bands, visualized through
staining or detecting radioactivity or fluorescence– Each band is a collection of DNA molecules of the
same length
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Video: Biotechnology Lab
Mixture of DNAfragments ofdifferent sizes
Completed gel
Longer(slower)molecules
Gel
Powersource
Shorter(faster)molecules
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