Chapter 20
Biotechnology
Unlocking Your Genome
https://www.cnbc.com/2015/12/10/unlocking-my-genome-was-it-worth-it.html
Concept 20.1 Introduction to Biotechnology
● Advances in DNA technology, methods to
manipulate DNA, have been made possible with innovations in accomplishing recombinant DNA, DNA molecules formed from combining two
different sources.
● Biotechnology is the manipulation of organisms or their components to make useful products, ie cheese, or wine.
● Genetic engineering is a particular type of
biotechnology that directly manipulates genes for practical purposes, ie transgenic organisms.
● Genetic engineering has sparked a revolution in
biotechnology and its potential applications are
being used in medicine and agriculture to criminal
justice and the study of cancer.
Concept 20.1 DNA Cloning Requires Tools
● To work directly with genes, scientists have developed methods to prepare small DNA segments into multiple copies through DNA cloning.
● Scientists use a plasmid, also known as a cloning vector, from the
bacterium E. coli to prepare multiple copies of a gene, gene cloning, with the use of restriction enzymes.
● Restriction enzymes or restriction endonucleases are naturally occurring bacterial enzymes discovered in the 1960s and have become quite useful in DNA cloning.
● Restriction enzymes look for a specific DNA sequence, generally 4 to 8 nucleotides in length, known as a restriction site and when found cut both DNA strands at precise points.
● The use of restriction enzymes on DNA create many cuts that produce restriction fragments, a single restriction enzyme when exposed to a restriction site will cut a DNA molecule in a reproducible way.
● The most useful restriction enzymes cut the DNA into a staggered manner creating sticky ends as opposed to blunt ends.
● DNA ligase seals the sugar phosphate bonds of the remaining DNA.
Concept 20.1 Cloning Genes in Bacterial Plasmids
● Recombinant plasmids or plasmids designed to carry foreign genes into E. coli are copied each time
E. coli reproduces through binary fission.● Gene cloning is a process that creates many copies of a gene of interest through the use of restriction enzymes and bacterial plasmids as outlined in the hummingbird example.
○ Plasmids are bioengineered with hummingbird DNA, restriction enzymes, and DNA ligase.
○ Restriction enzymes cut specific sequences in hummingbird DNA and bacterial plasmids.
○ DNA hummingbird fragments and cut plasmids are mixed together.
○ Mix recombinant E. coli with E. coli with mutant LacZ gene.
○ Grow bacteria in a medium with ampicillin and X-gal to identify colonies of E. coli with the hummingbird gene.
Concept 20.1 DNA Clones are Stored in Libraries?
● Genomic libraries are complete sets of plasmid-containing bacterial cell clones that carry a particular DNA segment from the original genome.
● Bacteriophages have been used as cloning vectors for making genomic libraries.
● Bacterial artificial chromosomes or BACs are used as vectors, they are large plasmids carrying just the genes of interest made of up 100-300kb.
● Clones are stored in multi-welled plates, one clone per well, ie plasmid with cloned beta-globin gene from the
hummingbird.
● Complementary DNA or cDNA is another type of DNA library created from fully processed mRNA and uses reverse transcriptase, DNA polymerase, and DNA ligase.
● Once cDNA is created it is modified by restriction enzymes
and inserted into a DNA vector forming a cDNA library.
Concept 20.1 Screening Libraries
● How do scientists detect the presence of a gene of interest in plasmids?
● Nucleic Acid hybridization is a process of complementary base pairing between the DNA of the gene of interest and its complementary single stranded molecule of either DNA or RNA that is called a nucleic acid probe, made of Each nucleic acid probe is made of radioactive isotopes.
● Once the gene of interest is found in a E. coli clone, those cells can be grown in large quantities for further study.
● Due to the complexity of eukaryotic gene expression, often scientists will often use an expression vector, which is a cloning vector with a highly active promoter upstream of the restriction site where the eukaryotic gene will be inserted.
● cDNA is used when introns are present in eukaryotic genes.
● Yeast, single-celled eukaryotic fungi are often used as cloning vectors because they contain plasmids, are eukaryotic, and are easily grown.
● DNA electroporation is another method to introduce recombinant DNA into
eukaryotic cells, creating pores in the membrane for DNA delivery.
Concept 20.1 Amplifying DNA through PCR
● When the source of DNA is scarce, ie at a crime scene, and large quantities of DNA are needed
polymerase chain reaction or PCR is used to make billions of copies of a targeted sequence in that DNA sample.
● PCR uses a 3-step cycle that starts a reaction that can produce an exponentially growing population of identical DNA molecules.
● Each cycle superheats the DNA sample to denature or separate DNA, and cools to allow annealing or complementary base pairing with DNA primers and nucleotides, the result after many cycles is lots of DNA!
● With each successive cycle the number of molecules
produced is 2
n, so after 30 cycles (n), a billion copies
are made.
Concept 20.2 DNA Technology Helps Study Genes
● Gel electrophoresis is a technique used to separate DNA fragments or proteins with the use of a gel, a liquid buffer, a gel chamber supplied with electrical current.
● DNA fragments or proteins separate along the gel surface, from the well they were injected into, based on molecular size and charge as they migrate along the gel surface from the anode (-) to cathode (+).
● The fragments, known as bands, are visible based on the dyes that are used in the process.
● One application of gel electrophoresis is restriction fragment analysis, in which DNA fragments created by restriction enzymes separate on the gel and form bands that can be compared in terms of migration distance, different size fragments based on nucleotide length (bp).
● Variations in DNA sequences called polymorphisms can alter fragment size after restriction enzyme digestions creating very predictable
fragments sizes or RFLPs.
● Southern blotting uses electrophoresis & nucleic acid hybridization to
search for specific bands for identification.
Concept 20.2 DNA Sequencing & Analyzing Gene Expression
● Today, DNA sequencing is automated by sequencing machines developed in the 1980s.
● “Next-generation sequencing” techniques of today synthesis one strand complementary strand of DNA that uses an
electronic monitor to identify each nucleotide letter (A,T,G,C) as its added one at a time.
● Northern blotting, similar to southern blotting, uses gel electrophoresis on mRNA which are later allowed to complementary base pair with nucleic acid probes.
● Reverse transcriptase-polymerase chain reaction or PT-PCR starts with mRNA that is transcribed into cDNA which serves as a template for PCR of the gene.
● In vitro mutagenesis uses specific mutations introduced to
cloned genes that can shed light on how it alters normal
phenotype expression, a form of gene function study.
Concept 20.3 Cloning and Stem Cells Research
● Organismal cloning, production of one or more organisms genetically identical to the “parent” that donated a single cell has great potential to generate stem cells.
● Totipotent cells, such as plant root cells, can dedifferentiate and give rise to other specialized cells.
● The nuclear potential of animal cells for cloning, unlike certain plant cells, is more restricted past embryonic development.
● In most nuclear transplantation cloning, only a small number of embryos develop normally to birth.
● The major goal of cloning human embryos is the production of stem cells, unspecialized cells that have the potential to
differentiate into specialized cells.
● Embryonic stem (ES) cells are pluripotent because they are capable of differentiating into many cell types.
● iPS or induced pluripotent stem cells are the result of
reprogramming differentiated cells into ES cells.
Concept 20.4 Practical Application of DNA Technology
● One important use of DNA technology is in the identification of human genes whose mutation plays a role in genetic disease or susceptibility non-genetic diseases.
● RT-PCR has been used to detect HIV RNA in blood or tissue samples.
● PCR is used to amplify DNA and then sequence the
disease-causing mutation, ie sickle cell disease, hemophilia.
● Identification of single nucleotide polymorphisms or SNPs associated with disease-causing alleles are used to test
patients for the abnormal allele.
● Gene therapy which is the introduction of healthy genes into afflicted individual’s cells has great potential to cure genetic diseases, prime candidates are stem cells.
○ Gene therapy raises technical issues and ethical issues.
