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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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CHAPTER 10 RECOMBINANT DNA

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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


  • To understand this chapter, you must also understand that

  • At present, sequencing technology is limiting, and genes cannot be sequenced or studied without many many copies of the same sequence.

  • Thus, considerable technical effort is devoted to getting many copies of one gene. The two processes used for this purpose are cloning and the polymerase chain reaction (PCR)


POLYMERASE CHAIN

REACTION

DIDEOXY SEQUENCING


“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

Serum

PCR

Sequencing

Bar code match


The fungal barcode gene

SSU

5.8S

LSU

Setosphaeria rostrata

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

“PCR”


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

primer

primer

72o copies DNA

DNA polymerase

Two identical copies


DNA sequencing


VII. DNA SEQUENCING

DNA SEQUENCING

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


DNA Sequencing can be done on a cloned gene or PCR product

AATCGGACTGGAGGCTTAGAACTGGATTT

PRIMER

C

Terminating fluorescently-labeled nucleotide

AATCGGACTGGAGGCTTAGAACTGGATTT

T

PRIMER

Terminating fluorescently-labeled nucleotide


DNA Sequencing can be done on a cloned gene or PCR product

3’TTAGCCTGACCTCCGAATCTTGACCTA

PRIMER

AATCGGACTGGAGGCTTAGAACTGGAT

AATCGGACTGGAGGCTTAGAACTGGATTT

AATCGGAC

AATCGGACTGGAGG

AATCGGACTGGAGGCTTAGAA


DNA Sequencing can be done on a cloned gene or PCR product

3’TTAGCCTGACCTCCGAATCTTGACCTA

PRIMER

3’T

3’TT

3’TTA

3’TTAG

3’TTAGC

3’TTAGCC

3’TTAGCCT


WELL

G

T

C

C

G

A

T

T

SEQUENCING GEL SEPARATES FRAGMENTS BY SIZE

An actual sequencing gel


DNA CLONING


DNA CLONING: THE PROCESS OF PLACING DNA FROM ONE ORGANISM INTO ANOTHER TO MAKE MULTIPLE COPIES

RESTRICTION ENZYMES ARE USED IN DNA CLONING


RESTRICTION ENZYMES

  • Restriction enzymes cut DNA at specific base sequences

  • Bacteria use them to defend themselves against infection

  • They are named for the bacteria that produce them

  • Eco R1 is derived from Escherichriacoli strain R-cuts palindrome GAATTC

  • HindIII is derived from Haemophilus influenza - cuts palindrome AAGCTT

  • “palindrome” – DNA sequence that is the same on both strands of DNA


CLONING CAN TAKE PLACE IN A NUMBER OF VECTORS

  • VECTOR: THE THING RECEIVING A FRAGMENT OF DNA, USUALLY A PLASMID OR VIRUS

  • MOST VECTORS HAVE BEEN GENETICALY MODIFIED TO RECEIVE DNA

  • MOST VECTORS HAVE BEEN MODIFIED SO THAT BACTERIA WITH A RECOMBINANT PLASMID CAN BE SELECTED FOR

    • -ANTIBIOTIC RESISTANCE

    • -COLOR SELECTION (BLUE WHITE COLORS)


LIBRARIES

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

  • Complementary DNA libraries

    • -derived from mRNA only

    • -represents only gene regions of a genome

    • -mRNA is isolated using its poly A tail and reverse-copied into DNA, then cloned.


FINDING A SPECIFIC GENE IN A LIBRARY

  • NUCLEIC ACID HYBRIDIZATION IS USED TO LOCATE SPECIFIC DNA SEGMENTS USING -

    • A GENE FROM A RELATED ORGANISM

    • mRNA FROM AN INDUCED CELL

    • ANTIBODIES CAN BE USED AS PROBES


known DNA sequence


BACTERIA CONTAINING CLONED GENES HAVE COMMERCIAL VALUE

HUMAN INSULIN

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

ETC


TRANSGENIC PLANTS

Methods


PLANTS CAN BE GENETICALLY MODIFIED

(SO CAN ANIMALS)

PLANTS WHICH HAVE RECEIVED A FOREIGN GENE ARE CALLED TRANSGENIC PLANTS

  • METHODS OF GETTING GENES INTO PLANTS TO MAKE TRANSGENIC PLANTS

    • AGROBACTERIUM

    • GENE GUN


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

protoplasts

callus

regeneration

Sugar cane plants

regeneration


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

Transform cells

Regenerate plants


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


TRANSGENIC PLANTS

Kinds of transgenic plants


Ethylene resistance

GeneticallyModified

normal

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

Not Sprayed

Sprayed


TRANSGENIC PLANTS

Opposition to transgenics


Protests against transgenic plants


END


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