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Restriction enzymes come from bacteria and recognize a particular pattern of DNA, often 4, 6 or 8 base pairs long, and then cut the DNA within this recognized sequence.
Bacteria use these enzymes to kill off other competing bacteria by cutting up their DNA.
How do they cut? STICKY ENDS B LUNT ENDS
ACT GAA TTC CGG AAT GAA TTC
TGA CTT AAG GCC TTA CTT AAG
Where would the enzyme EcoRI cut?
ACT GAA TTC CGG AAT GAA TTC
TGA CTT AAG GCC TTA CTT AAG
There would be three pieces: one 4 bases, one 12 bases, and one 5 bases .
Making recombinant DNA in plasmids
The collection of thousands of clones of bacteria containing recombinant plasmids is called a genomic library.
Genes can be cloned into vectors such as plasmids
Fig. 20-2 DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA ( plasmid ) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Plasmid Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth 2 4 1 3
Fig. 20-2a DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA (plasmid) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Plasmid 2 1 2
Fig. 20-2b Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein 4 Recombinant bacterium Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth 3
Plasmid and human DNA are isolated.
Both DNAs are cut with the same restriction enzyme.
“ new” DNA is ligated into plasmid
Recombinant plasmids are inserted into bacterial cells.
Plate bacteria on agar. Bacteria will express new genes.
Nucleic Acid Hybridization
Used to detect genes
The DNA of the cell is denatured to produce single stranded DNA.
The radioactive probe will hybridize (bond) with complementary bases if present.
Probes can be radioactive isotopes or
The radioactive probe is made by determining a short segment of the protein sequence, then "back translating" to the possible DNA sequences. Short DNA sequences are synthesized to match the protein sequence. Then these DNA oligomers (known as "oligos") are radiolabeled, and applied to the blotted clones. They should hybridize only to clones containing sequence encoding the desired protein.
Expression of eukaryotic genes in prokaryotes
Use an expression vector with a prokaryotic promoter upstream from the location of the gene.
Create artificial genes without introns since bacteria do not have the machinery for eliminating introns.
What are YACS?
Yeast artificial chromosomes that carry foreign DNA.
Yeast cells have plasmids that can act as vectors.
In PCR, a heat-stable DNA polymerase is used, most commonly Taq Polymerase from the thermophilic microbe Thermus aquaticus. Thomas Brock discovered T. aquaticus from a hot spring at Yellowstone National Park.
Why is PCR used prior to cloning a gene in cells?
The task of later identifying the clone carrying the gene is simplified.
Applications of PCR
PCR has replaced cloning for many purposes, particularly the sequencing of DNA.
It is faster and requires no vectors, which can mutate as they reproduce.
It can be used forensically, to amplify tiny amounts of DNA from criminal evidence; or clinically, to detect DNA sequences linked to inherited disorders.
What is gel electrophoresis?
A technique to separate DNA based on the movement of DNA fragments from neg to pos (DNA is neg).
Smaller fragments travel farther.
Samples are placed in gels.
Southern Blotting DNA Fingerprinting
Cut DNA into fragments with restriction enzymes.
Blot onto nylon membrane.
Apply radioactive probes.
Wash to remove unbonded probes.
Used to sequence short segments of DNA
Fragments are incubated with fluorescent dyes.
When fragments hybridize with the tagged nucleotide, the hybridization stops.
Fragments are electrophoresed and analyzed.
Early DNA Sequencing
Analyzing Expression of Genes
Northern Blotting , in situ hybridization – using radioactive probes to look for mRNA being produced
RT-PCR – Reverse transcriptase-polylmerase chain reaction – makes cDNA from mRNAs and then PCRs the DNA for electrophoresis
Micro – arrays - Isolate mRNA from cells, make cDNA using reverse transcriptase, then uses cDNA to explore collections of genomic DNA
Microarrays are useful in discerning gene expression in different tissues AND at different stages of development.
Different brightness and
colors signify rates of
Determining Gene Function
In vitro mutagenesis – changes made to cloned gene, gene returned to cell and it “knocks out” the normal gene. Then look for abnormalities.
RNA interference (RNAi) – uses double RNA to block translation of mRNA.
Organismal cloning – producing genetically identical individuals from a single somatic cell of a multicellular organism
Steward demonstrated genomic equivalence in plants by growing carrot plants from differentiated root cells.
Most plant cells remain totipotent , retaining the ability to give rise to a complete new organisms.
Briggs and all transplanted nuclei from embryonic frog cells into enucleated egg cells and produced cloned frogs
Nuclear transplantation – name of process
Whether normal development occurred depended on developmental age of the transplanted nucleus.
Fig. 20-17 EXPERIMENT Less differ- entiated cell RESULTS Frog embryo Frog egg cell UV Donor nucleus trans- planted Frog tadpole Enucleated egg cell Egg with donor nucleus activated to begin development Fully differ- entiated (intestinal) cell Donor nucleus trans- planted Most develop into tadpoles Most stop developing before tadpole stage
And then Dolly came along in 1997
Fig. 20-18 TECHNIQUE Mammary cell donor RESULTS Surrogate mother Nucleus from mammary cell Cultured mammary cells Implanted in uterus of a third sheep Early embryo Nucleus removed Egg cell donor Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor Egg cell from ovary Cells fused Grown in culture 1 3 3 4 5 6 2
Why Dolly died young 6 yrs
Dolly's telomeres were found to be approximately 80% of the length they should be for a sheep her age.
Also there is the concern of damaged DNA being carried into the clone
Cloned animals do not look exactly like the transplanted nucleus due to cytoplasmic affects.
Rainbow CC CC and her Surrogate mom
In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth
Many epigenetic changes , such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
Relatively unspecialized cells that continue to reproduce themselves and can be induced to form specialized cells
Embryonic cells are more totipotent than adult stem cells
Therapeutic cloning – using stem cells to replace organs and tissues
Reproductive cloning – using stem cells to reproduce new organisms
Both raise ethical
Benefits of DNA technology
identification of human genes in which mutation plays a role in genetic diseases
Single nucleotide polymorphisms (SNPs ) are useful genetic markers
These are single base-pair sites that vary in a population
When a restriction enzyme is added, SNPs result in DNA fragments with different lengths, or restriction fragment length polymorphisms ( RFLP)
Fig. 20-21 Disease-causing allele DNA SNP Normal allele T C
Human Gene Therapy
Gene therapy is the alteration of an afflicted individual’s genes
Vectors, such as viruses, are used for delivery of genes into specific types of cells, for example bone marrow
It may be difficult to target cells.
Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations
Fig. 20-22 Bone marrow Cloned gene Bone marrow cell from patient Insert RNA version of normal allele into retrovirus. Retrovirus capsid Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Inject engineered cells into patient. 1 2 3 4
Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases
In particular “ pharm ” animals and plants can be used to produce certain products
Forensic Evidence and Genetic Profiles
An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids
Even more sensitive is the use of genetic markers called short tandem repeats (STRs ), which are variations in the number of repeats of specific DNA sequences
Fig. 20-24 This photo shows Earl Washington just before his release in 2001, after 17 years in prison. These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. (a) Semen on victim Earl Washington Source of sample Kenneth Tinsley STR marker 1 STR marker 2 STR marker 3 (b) 17, 19 16, 18 17, 19 13, 16 12, 12 14, 15 11, 12 13, 16 12, 12
Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels
Genetic Engineering in Plants
Agricultural scientists have endowed a number of crop plants with genes for desirable traits
The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives
Fig. 20-25 Site where restriction enzyme cuts T DNA Plant with new trait Ti plasmid Agrobacterium tumefaciens DNA with the gene of interest Recombinant Ti plasmid TECHNIQUE RESULTS