CHAPTER 10 RECOMBINANT DNA. To understand this chapter, you must understand that The DNA double helix can be separated into two single helices by heat or chemical treatment (usually 95 C) When DNA is cooled, DNA sequences that are similar or the same will anneal (zip together). 50-60C. 95C.
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“The fungus behind the outbreaks was initially identified as Aspergillus, but as more testing of patients has been completed, it's become clear that another fungus — a kind of black mold called Exserohilum (Setosphaeria) — is the primary cause. CDC's fungal disease laboratory confirmed Exserohilum in 10 people with meningitis and Aspergillus in just one.” 195 affected/19 dead
Bar code match
The fungal barcode gene
Eukaryota; Fungi; Dikarya; Ascomycota; Pezizomycotina; Dothideomycetes; Pleosporomycetidae; Pleosporales; Pleosporineae; Pleosporaceae; Setosphaeria
agaaaaatat gagggtgtgg tttgctggga acagcgtccg ccgcaggtat ttttcagcca gtgtctgttg cgcacttttt gtttcctggg cgagttcgct cgccaccagg acccaaccat aaaccttttt ttatgcagtt gcaatcagcg tcagtataat aattcaattt attaaaactt tcaacaacgg atctcttggt tctggcatcg atgaagaacg cagcgaaatg cgatacgtag tgtgaattgc agaattcagt gaatcatcga atctttgaac gcacattgcg ccctttggta ttccaaaggg catgcctgtt cgagcgtcat ttgtaccctc aagctttgct tggtgttggg cgtctttttg tctctcccct tgttggggga gactcgcctt aaaacgattg gcagccgacc tactggtttt cggagcgcag cacaaatttg cgccttccaa tccacggggc ggcatccagc aagcctttgt tttctataac aaatccacat tttgacctcg gatcaggtag ggatacccgc tgaacttaag catatcaata a
THE POLYMERASE CHAIN REACTION
GENES CAN BE COPIED BY PCR
Uses alternating temperatures and primers specific to a gene region to make millions of copies of a gene for sequencing or study
95o separates DNA strands
52o anneals primers
72o copies DNA
Two identical copies
DNA sequencing is a procedure in which the sequence of bases in a gene is determined.
The most common method of DNA sequencing (Sanger sequencing) uses a one-directional PCR process and fluorescently-labeled dideoxy nucleotides (dideoxy nucleotides lack an oxygen on the number 3 carbon of ribose and cannot add a further nucleotide to a growing DNA strand)
Newer rapid methods of DNA sequencing have been and are being developed
Terminating fluorescently-labeled nucleotide
Terminating fluorescently-labeled nucleotide
SEQUENCING GEL SEPARATES FRAGMENTS BY SIZE
An actual sequencing gel
DNA CLONING: THE PROCESS OF PLACING DNA FROM ONE ORGANISM INTO ANOTHER TO MAKE MULTIPLE COPIES
RESTRICTION ENZYMES ARE USED IN DNA CLONING
CLONING CAN TAKE PLACE IN A NUMBER OF VECTORS
LIBRARIES ARE A COLLECTION OF CLONED DNA FRAGMENTS
EACH FRAGMENT IS IN A DIFFERENT BACTERIAL OR VIRAL CELL
THE COLLECTION OF CELLS MAKES UP THE LIBRARY (e.g., a rice genomic library)
THERE ARE MANY DIFFERENT KINDS OF LIBRARIES
FINDING A SPECIFIC GENE IN A LIBRARY
known DNA sequence
BACTERIA CONTAINING CLONED GENES HAVE COMMERCIAL VALUE
HUMAN GROWTH HORMONE
ICE-MINUS PSEUDOMONAS BACTERIA
DIAGNOSTIC TOOLS (BRAC1)
BACTERIA CONTAINING CLONED GENES HAVE SCIENTIFIC VALUE
CLONED CHLOROPLAST GENES (CLEAN ENERGY)
CLONED GENES FOR PLANT DISEASE RESISTANCE
PLANTS CAN BE GENETICALLY MODIFIED
(SO CAN ANIMALS)
PLANTS WHICH HAVE RECEIVED A FOREIGN GENE ARE CALLED TRANSGENIC PLANTS
TRANSGENIC PLANTS MAY BE CREATED USING AGROBACTERIUM TUMIFACIENS, A NATURAL VECTOR
Many plant cells are “totipotent”, i.e., a single cell has the capacity to regenerate a complete plant
Sugar cane plants
Some transgenic plants are made by infecting the plant with a bacterium, Agrobacterium tumefaciens which contains a plasmid. The plasmid integrates into the plant DNA causing a gall
Agrobacterium tumifaciens or crown gall disease
insert a gene
TRANSGENIC PLANTS MAY BE CREATED USING A GENE GUN
Some transgenic plants are made by shooting DNA-coated particles into tissues and regenerating plants from the tissues
Kinds of transgenic plants
Contains a mutant ethylene receptor gene – delayed ripening. 100 days after picking
The yellow color of this genetically modified rice is due to its ability to make Beta-carotene. It also contains a gene for a bean iron storage protein, ferritin. The modified rice will provide vitamin A and iron. 400 million people worldwide are deficient in vitamin A leading to illness and blindness. Iron is the number one micronutrient deficiency.
Novartis, Astra-Zeneca and Monsanto are claiming exclusive ownership to the basic patents related to rice research. Further, neither Monsanto nor Astra - Zeneca said they will give up their patents on rice – they merely gave royalty free licenses to public sector scientists for development of "golden rice".
Science 285:994 (Aug 1999)
Bacillus thuringiensis (BT) toxin
Upon sporulation, B. thuringiensis forms crystals of insecticidal δ-endotoxins (called crystal proteins or Cry proteins). These attach to insect gut walls and create a hole which kills the insect.
Cry toxins have specific activities against moths and butterflies, flies and mosquitoes, beetles, wasps, bees, ants, sawflies, and nematodes
European corn borer-resistant transgenic plants containing BT toxin developed by Mycogen.
Transgenic Insect-resistant cotton (Monsanto).
Plants contain BT toxin
Glyphosate (Roundup) resistant corn sprayed with herbicide to kill weeds (Monsanto). The gene has escaped and created super weeds as predicted
Opposition to transgenics
Protests against transgenic plants