Notes

College Biology Chapter 20

Recombinant DNA technology: the use of laboratory techniques to isolate and manipulate fragments of DNA- to benefit humans.

Any DNA molecule that has been manipulated so it contains DNA from two or more sources (recombinant).

READ about insulin and recombinant DNA (p.411) and continue (p.421).

Gene cloning: the recombinant molecules are replicated to produce many identical copies.

*DNA for a specific gene

Genomics: the molecular analysis of the entire genome of a species. Help us understand differences of normal cells vs. cancerous cells.

Biotechnology: the use of living organism o the products of living organism for human benefit. Ex. bacteria making human insulin, crops produce insecticides, farm animals make human medicine.

Genetic engineering: the direct manipulation of genes for practical applications.(p.412)

Vector: the DNA that acts as a carrier of DNA for the segment about to be cloned.

Plasmids: small circular viruses.

Viral vector: an alternative type of vector.

*Viruses can be used as vectors to carry pieces of DNA

Restriction enzymes: an enzyme that recognizes particular DNA sequences and cleaves the DNA backbone at two sites.

*These are naturally made by bacteria to cut viral DNA as a protection method.

* Discovered by Werner Ariber, Hamilton Smith, and Daniel Nathans in the 1960s and 70s.

Restriction sites: the sequences recognized by restriction enzymes.

Palindromic – The DNA sequence is identical when read in the opposite direction in the complementary strand.

p. 413 Table 20.1

Sticky ends: DNA fragments that have a single stranded ends that will hydrogen bond to other DNA that are cut with the same enzyme due to their complementary strand.

Recircularized vector – The two ends of the vector that are cut ligate (connect) back together

Recombinant vector: a hybrid vector containing a piece of chromosomal DNA.

Selectable marker: a gene whose presence can allow organisms (such as bacteria) to grow under a certain set of conditions.

 Ex. the presence of an antibiotic selects for the growth of cells expressing the amp8 gene.

*This gene degrades antibiotics.

*Dyes can be used to separate the bacterial colonies with recircularized vectors from recombinant vectors. P. 414 Fig. 20.3

DNA library: a collection of vectors, usually within bacterial cells.

*Many different DNA fragments are formed with restriction wnzymes.

Genomic library: inserts are derived from chromosomal DNA.

Complementary DNA:cDNA: DNA molecules that are made up of mRNA as a starting material. mRNA works better because there are no introns.

cDNA library: a collection of recombinant vectors that have cDNA inserts.

Gel electrophoresis: a technique that is used to separate macromolecules on a gel (ex. protein and DNA).

*This method separates the DNA fragments by size/length, charge, or mass.

*Separate bands develop over time, which can be stained and identified.

Polymerase chain: PCR: many copies of DNA in a defined region, perhaps accompanying a gene or part of a gene.

Process:

1)      Denature the double-stranded temple DNA with heat to make single strands.

2)      Add both reverse primer and forward primers.

3)      Taq polymerase uses dNTPs to catalyze the synthesis of the complementary DNA.

(denature – anneal – extension)

Annealing – the binding of a primer to a specific site on the DNA strand.

Thermocycler – a device that controls temperature and automates the timing of each step.

20.2 Genomics

Functional genomics: studies the expression of a genome.

Ex. which genes are turned on or off in normal cells vs. cancer cells.

Bacterial artificial chromosome: BAC: a cloning vector derived from F factors that can contain large DNA inserts.

*Normal plasmids can only insert a few thousand before deletion or insertion.

YAC: Yeast Artificial Chromosome: a cloning vector that can handle several hundred thousand nucleotides.

Mapping: process of determining the relative locations of genes or other DNA segments along a chromosome.

Contig: a contiguous region of a chromosome that is found overlapping regions within a group of vectors.

DNA sequencing: a method to determine the base sequence of DNA.

*This has been vital in our understanding of human diseases.

Dideoxy chain-termination method: (dideoxy sequencing):the most commonly used method of DNA sequencing. Method developed by Frederick Sanger in 1977. He reasoned that dideoxynucleotide is added, the DNA will no longer grow.

Pyrosequencing – a new method of DNA sequencing that is based on the detection of released pyrophosphate during DNA synthesis.

DNA microarray: used to monitor the expression of thousands of genes simultaneously.

20.3 Biotechnology

Biotechnology – The use of living organisms, on products from living organisms, as a way to benefit humans.

* In the 1900s, microorganisms were used to treat sewage. (p. 421)

Bioremediation: use of living organisms, typically microorganisms or plants, to detoxify pollutants in the environment.

Transgenic: used to describe an organism that carries genes that were introduced using molecular techniques such as gene cloning.

Transgenic organisms: Genetically modified organisms (GMOs) – organisms that have a new gene inserted into their DNA.

* Transgenic fish, sheep, pigs, goats, and cattle are being researched.

A)      Gene addition:  the insertion of the cloned gene into the genome.

B)      Gene replacement: cloned genes that recombine with the normal gene on a chromosome.

*Cystic fibrosis (cF) defective gene has been identified and scientists may be able to remove the cF gene from a person’s somatic cells.

Gene knockout: an organism in which both copies of a functional gene have been replaced with non-functional copies. Experimentally, this can occur via gene replacement. (p. 423)

Molecular pharming: production of medically important proteins in the mammary glands of livestock. (p. 423 Fig. 20.11)

Ex. factor IX to treat hemophilia, tissue plasminogen activator (tpa) to solve blood clots, and alpha-1-antitrypsin for treatment of emphysema.

Molecular pharming is better than using bacteria because:

A)      Proteins function more properly in humans when made by mammals.

B)      Proteins may degrade quickly or fold improperly in bacteria.

C)      Protein yield from milk is much faster than in bacteria.

Totipotent – an entire organism can be regenerated from somatic cells. Most plants exhibit this.

Ti plasmid: tumor – inducing plasmid from the bacterium Agrobacterium tumefaciens. (p. 424 Fig. 20.12) *Transgenic research has sought to produce plant strains that are resistant to insects, disease, and herbicides.

Reproductive cloning: cloning of multicellular organisms. (p. 426 halfway down) Cloning may make organisms susceptible. Ex. B+ corn p. 425

Bacillas thuringienis (B+) is a bacterium that produces a toxin that kills caterpillars and beetles which were used in insecticides for decades. Researchers have succeeded in cloning the toxin genes from the B+ bacterium and transferring the genes into the corn plant.

*Concerns are:

A)      B+ corn kills other insects (pollinators)

B)      Insects may become resistant

C)      People may become allergic to the plants

D)      People may consume more toxins.

DNA fingerprinting: technology that can identify and distinguish among individuals based on variations in their DNA.

Short tandem repeat sequences: STRs: short DNA sequences that are repeated many times in a row. These noncoding regions of DNA are specific to species and individuals.

Gene therapy – introduction of cloned genes into living cells in an attempt to cure diseases.