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Page 1: 11 9 09 Lecture Slides

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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Page 3: 11 9 09 Lecture Slides

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

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Sugar(deoxyribose)

Thymine (T)

Nitrogenous base(A, G, C, or T)

Phosphategroup

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Pyrimidines

Guanine (G)Adenine (A)Cytosine (C)Thymine (T)

Purines

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Sugar(ribose)

Uracil (U)

Nitrogenous base(A, G, C, or U)

Phosphategroup

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Ribose

Cytosine

Uracil

Phosphate

Guanine

Adenine

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Hydrogen bond

Basepair

Partial chemical structure Computer modelRibbon model

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Basepair

Ribbon model

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Hydrogen bond

Partial chemical structure

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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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Page 14: 11 9 09 Lecture Slides

Cytoplasm

Nucleus

DNA

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Cytoplasm

Nucleus

DNA

Transcription

RNA

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Cytoplasm

Nucleus

DNA

Transcription

RNA

Translation

Protein

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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

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Polypeptide

Translation

Transcription

Gene 1

DNA molecule

DNA strand

Codon

Amino acid

Gene 2

Gene 3

RNA

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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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Page 22: 11 9 09 Lecture Slides

– 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

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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

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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

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E. coli bacteriumPlasmid

Bacterialchromosome

Gene of interestDNA

Cell with DNAcontaining geneof interest

Isolateplasmid

IsolateDNA

1

2

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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

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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

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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

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RecombinantDNAplasmid

Geneof interest

Recombinantbacterium

Put plasmidinto bacteriumby transformation

7

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RecombinantDNAplasmid

Geneof interest

Recombinantbacterium

Cloneof cells

Put plasmidinto bacteriumby transformation

Allow bacteriumto reproduce

8

7

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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

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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

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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

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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

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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

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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

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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

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Cycle 3yields 8 molecules

Cycle 2yields 4 molecules

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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

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Mixture of DNAfragments ofdifferent sizes

Completed gel

Longer(slower)molecules

Gel

Powersource

Shorter(faster)molecules