Chapter 12 DNA Technology and Genomics
A. Plasmids
Bacterial origin: circular DNA sequence: not associated with the bacterial chromosome
Recombinant DNA technology to give:
1) Therapeutic effect
2) provide “better” resistance to pests
3) clean up oil spills
The little factory that could …
1) isolate the plasmid from bacterium
2) Grab DNA with gene of interest from host cell (e.g., human)
3) splice that gene with the plasmid -> this will produce recombinant DNA
4) bacterial cell will take up this plasmid (with the foreign) DNA through the process of bacterial transformation = call this bacterium: recombinant bacterium
5) recombinant bacterium will reproduce gene products through transcription of recombinant DNA and translation of the resulting mRNA into a gene product (polypeptide)
Genetic engineering
B. Enzymes to cut and paste DNA
-Restriction enzyme: recognize a particular sequence
-And then cutting of DNA into fragments
Fragments will contain a sticking end:TTAA
-Add another DNA fragment from another source: “glue” together
-DNA ligase: “pastes” together the fragments, the foreign DNA to produce the recombinant DNA molecule
C. Genes can be cloned in recombinant plasmids
1. isolate DNA from two sources
2. cut both DNAs with same restriction enzyme
3. Mix DNAs together; let them base pair and
4. add DNA ligase to bond the DNA covalently
5. form recombinant DNA and then place the plasmid into the bacterium via transformation
6. clone the bacteria
7. isolate gene product
D. Cloned Genes can be stored in genomic libraries
BIOINFORMATICS
Store these cloned genes in genomic library
Collections of phage clones (viruses) and of bacterial clones
E. Reverse transcriptase helps make genes for cloning
1. chosen cells transcribe their genes into mRNA
2. researcher isolate the mRNA and make single-stranded DNA, usng the enzyme reverse transcriptase
3. these enzymes will break down mRNA
4. DNA polymerase to synthesis the second strand
This procedure allows the synthesis of a gene WITHOUT introns! This means we have a gene that can be immediately expressed.
In nature, DNA contains genes with Introns and Exons
F. Recombinant cells and organisms can mass-produce gene products
List of products made with recombinant DNA technology
Human insulin
Human growth hormone
Epidermal growth factor
Interleukin-2
Bovine growth hormone
Cellulase
Taxol
Interferons (alpha and gamma)
Hepatitis B vaccine
Erythropoietin (EPO)
Factor VIII
Tissue plasminogen activator
G. DNA technology is changing the pharmaceutical industry and medicine
Pharmaceuticals, vaccines, hormones
H. Nucleic acid probes identify clones carrying specific genes
Nucleic acid probe will be able to recognize certain nucleotide sequences
Trace genetic ancestry
I. DNA microarrays test for the expression of many genes at once
DNA microarray test: special wells with treated DNA that fluoresces
Used to test for toxicity, disease mechanisms, patterns of gene expression in tumors
J. Gel electrophoresis sorts DNA molecules by size
Electrophoresis to separate DNA molecules by their molecular weight
Long molecules: high molecule weight
Short molecules: lower molecular weight
Applying electricity to the molecule and the molecule will move to one end of the device based on its molecular weight
DNA fingerprinting
K. Restriction fragment length polymorphisms can be used to detect differences in DNA sequences
Number of restriction fragments and their size reflect the specific sequence of nucleotides in the starting DNA
Any differences in these restriction fragments are called RFLPs (restriction fragment length polymorphisms)
Take DNA from chromosomes – cut them up with the restriction enzyme
And then compare the fragments (long ones and the short ones) and check for commonality
1) crime scene and the suspect
2) Check out for harmful alleles
Dominant
Recessive
Check out use of recombinant DNA technology to “cure” sickle cell anemia!
L. DNA technology is used in courts of law
Can free prisoners based on DNA evidence
Can imprison defendents
M. Gene therapy may someday help treat a variety of diseases
Bone marrow transplants – save people who have leukemia
In vitro gene therapy
Cystic fibrosis – can use spray
Ex vivo gene therapy
N. PCR method is used to amplify DNA sequences
PCR: Polymerase chain reaction
Allows for replication of DNA sequences
Used for DNA fingerprinting, especially from very old samples and from
samples that are very small
O. Human Genome Project is an application of DNA technology
1. genetic linkage mapping – get markers
2. physical mapping – number of base pairs between markers
3. DNA sequencing
Map of the human genome
P. Most of the human genome does not consist of genes???
Non-coding DNA versus coding DNA
Repetitive DNA: nucleotide sequences present in many copies
Telomeres: repetitive DNA at chromosome ends
If lose telomeric DNA -> can lead to cell death
Transposons: DNA segments that can move or be copied from one location to another in a chromosome or between chromosomes
Q. Science of genomics compares whole genomes
Genomics: whole sets of genes
Humans: 2.9 billion base pairs and 25,000 genes
Proteomics: full protein sets
Catalog life on a global perspective?
R. Genetically modified (GM) organisms are transforming agriculture
GM organisms: have acquired one or more genes by artificial means rather than through traditional breeding methods
Plasmid – bacterial DNA
Transgenic plants
Transgenic animals
S. Could GM organisms harm human health or the environment?
Genetically Modified ….
Add genes for herbicide resistance??