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DNA Technology and the Human Genome. Chapter 12. Bacteria as Tools for Manipulating DNA. DNA Technology. The Human Genome Project Better understand diseases and their causes Gene therapy The production of vaccines, cancer drugs, and pesticides

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dna technology
DNA Technology
  • The Human Genome Project
    • Better understand diseases and their causes
    • Gene therapy
  • The production of vaccines, cancer drugs, and pesticides
  • Engineered bacteria that can clean up toxic wastes
    • Restore damaged ecosystems
bacteria as a tool for manipulating dna

DNA enterscell

Fragment ofDNA from anotherbacterial cell

Bacterial chromosome(DNA)

Bacteria as a Tool for Manipulating DNA
  • In nature, bacteria can transfer DNA in three ways
  • Transformation, the taking up of DNA from the fluid surrounding the cell
    • Can even take up DNA from dead cells
bacteria as a tool for manipulating dna1
Conjugation, the union of cells and the DNA transfer between them

Transduction, the transfer of bacterial genes by a phage

Mating bridge

Phage

Fragment ofDNA from anotherbacterial cell(former phagehost)

Donor cell(“male”)

Recipient cell(“female”)

Figure 12.1B

Bacteria as a Tool for Manipulating DNA
slide7

Donated DNA

Degraded DNA

Crossovers

Recipient cell’schromosome

Recombinantchromosome

  • The transferred DNA is then integrated into the recipient cell’s chromosome
bacterial phages as carriers
Bacterial plasmids can serve as carriers for gene transfer

An F factor is a DNA segment in bacteria that enables conjugation and contains an origin of replication

The F factor starts replication and transfers part of the chromosome

F factor (integrated)

Male (donor) cell

Origin of F replication

Bacterial chromosome

F factor startsreplication andtransfer of chromosome

Recipient cell

Only part of thechromosome transfers

Recombination can occur

Bacterial Phages as Carriers
slide9
An F factor can exist as a plasmid, a small circular DNA molecule separate from the bacterial chromosome

R plasmids carry genes for resistance of antibiotics and that is how bacteria can become resistant

F factor (plasmid)

Male (donor) cell

Bacterial chromosome

F factor startsreplication andtransfer

Plasmids

Plasmid completestransfer andcircularizes

Cell now male

plasmids are used to customize bacteria
Plasmids are used to customize bacteria
  • Plasmids are key tools for DNA technology
    • Researchers use plasmids to insert genes into bacteria
    • Plasmids are obtained from other bacteria
    • Desired DNA inserted into plasmid
    • Bacteria takes up DNA
  • Can be used for several applications
slide11

Cell containing geneof interest

1

Bacterium

Plasmidisolated

2

DNA

isolated

3

Gene inserted into plasmid

Bacterialchromosome

Plasmid

Gene ofinterest

Recombinant DNA(plasmid)

DNA

4

Plasmid put intobacterial cell

Recombinantbacterium

5

Cell multiplies withgene of interest

Copies of gene

Copies of protein

Gene for pestresistanceinserted intoplants

Clones of cell

Protein used to make snow format highertemperature

Gene used to alter bacteriafor cleaning up toxic waste

Protein used to dissolve bloodclots in heart attack therapy

how they insert dna into plasmids

Restriction enzymerecognition sequence

1

DNA

Restriction enzymecuts the DNA intofragments

Restriction enzymecuts the DNA intofragments

2

Sticky end

Addition of a DNAfragment fromanother source

3

Two (or more)fragments sticktogether bybase-pairing

4

DNA ligasepastes the strand

5

How they insert DNA into plasmids
  • Enzymes are used to “cut and paste” DNA
  • Restriction enzymes cut DNA at specific points
    • Recognize specific sequences
    • Make ‘sticky ends’
  • DNA ligase “pastes” the DNA fragments together
    • Catalyst for hydrogen bonds
  • The result is recombinant DNA
cloning genes using recombinant plasmids

1

Isolate DNAfrom two sources

Human cell

E. coli

2

Cut both DNAs with the same restriction

enzyme

Plasmid

DNA

Gene V

Sticky ends

3

Mix the DNAs; they joinby base-pairing

4

Add DNA ligaseto bond the DNA covalently

Recombinant DNAplasmid

Gene V

5

Put plasmid into bacteriumby transformation

6

Clone the bacterium

Cloning genes using recombinant plasmids
  • Bacteria take the recombinant plasmids and reproduce
  • This clones the plasmids and the genes they carry
    • Products of the gene can then be harvested
  • Make large amounts of a desired gene so that its protein can be manufactured
genomic libraries

Genome cut up with restriction enzyme

Recombinantplasmid

Recombinantphage DNA

OR

Phage

clone

Bacterialclone

Phage

library

Plasmid

library

Genomic Libraries
  • Recombinant DNA

technology allows the construction of genomic libraries

    • Genomic libraries

are sets of DNA

fragments containing

all of an organism’s genes

  • Copies of DNA fragments can be stored in a cloned bacterial plasmid or phage
using mrna as a template

CELL NUCLEUS

Exon

Intron

Exon

Intron

Exon

DNA ofeukaryoticgene

Transcription

1

RNA

transcript

RNA splicing(removes introns)

2

mRNA

Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand

3

TEST TUBE

Reverse transcriptase

Breakdown of RNA

4

cDNA strand

Synthesis of secondDNA strand

5

cDNA of gene(no introns)

Using mRNA as a Template
  • Researchers can use mRNA as a template to isolate the gene that makes that mRNA
  • Reverse transcriptase can be used to make smaller cDNA libraries
    • These contain only the genes that are transcribed by a particular type of cell
nucleic acid probes

Radioactiveprobe (DNA)

Mix with single-stranded DNA fromvarious bacterial(or phage) clones

Single-strandedDNA

Nucleic Acid Probes
  • In order to find the bacteria or phage that contains the desired gene in a library
  • Radioactive probes of complimentary DNA sequences to the desired gene can be used to find the desired gene
slide18

Bacterial colonies containingcloned segments of foreign DNA

Radioactive DNA

Transfercells tofilter

1

Solutioncontainingprobe

Filterpaper

Treat cellson filter toseparateDNA strands

Add probeto filter

ProbeDNA

2

3

Gene ofinterest

Single-strandedDNA from cell

Hydrogen-bonding

Autoradiography

4

Colonies of livingcells containinggene of interest

Developed film

Compare autoradiographwith master plate

5

Master plate

  • Colonies are blotted on filter paper
  • Paper is treated to break up DNA
  • Probe is added
  • Paper laid on photographic film
  • Film is compared to colonies
  • Gene is identified
gel electrophoresis
Gel Electrophoresis
  • Physically sorts out macromolecules (DNA, RNA) on the basis of their charge and size
  • Current is run through the gel and since DNA is negatively charged it moves through the gel
  • The longer the DNA molecules are. The slower they move
  • Bands are made, each consisting of DNA molecules of one size
gel electrophoresis1

Mixture of DNAmolecules ofdifferent sizes

Longermolecules

Powersource

Gel

Shortermolecules

Glassplates

Completed gel

Gel Electrophoresis
  • Restriction fragments of DNA can be sorted by size
restriction fragment analysis
Restriction Fragment Analysis
  • Everyone’s DNA sequence is different
  • Scientists can compare DNA sequences of different individuals based on the size of the fragments created by restriction enzymes
  • They can only use DNA that varies from person to person
  • When run on a gel it makes a distinct pattern
restriction fragment analysis1

1

2

Allele 1

Allele 2

w

Longer fragments

Cut

z

x

Shorter fragments

Cut

Cut

y

y

DNA from chromosomes

Restriction Fragment Analysis
detecting harmful alleles

Restriction fragmentpreparation

1

Restrictionfragments

Gel electrophoresis

2

Filter paper

Blotting

3

Radioactive probe

4

Probe

Detection of radioactivity(autoradiography)

5

Film

Detecting Harmful Alleles
  • Radioactive single-stranded DNA complimentary strands are used to verify the presence of certain nucleic acid sequences known to code for harmful alleles
slide24
PCR
  • The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube
  • DNA sample mixed with DNA polymerase, nucleotides and other things and it replicates exponentially
  • Can replicate billions of clones within a few hours
slide25

InitialDNAsegment

1

2

4

8

Number of DNA molecules

PCR
  • Can copy a specific segment of DNA in a mass of DNA
  • Needs only minute amounts of DNA
  • Cannot produce large amounts of DNA
  • Scientists are using it to identify missing cases with bone marrow, prehistoric beasts etc
the human genome
The Human Genome
  • The 23 chromosomes in the haploid human genome contain about 3 billion nucleotide pairs
    • This DNA is believed to include about 35,000 genes and a huge amount of noncoding DNA (do not code for proteins)
      • About 97% of the total human genome
  • Much of the DNA between genes consists of repetitive DNA
    • Small sequences repeated over and over again on the same chromosome
    • Large sequences that are scattered throughout the genome
the human genome project
The Human Genome Project
  • The Human Genome Project involves:
    • Genetic and physical mapping of chromosomes
      • Gene mapping and fragment analysis
    • DNA sequencing
      • Nucleotide sequence of the genes
    • Comparison of human genes with those of other species
      • Help the scientists interpret the human data
dna technology and the criminal court

Defendant’sblood

Blood fromdefendant’sclothes

Victim’sblood

DNA Technology and The Criminal Court
  • Restriction fragment analysis using gel electrophoresis
    • Compare DNA from a crime

scene to a

sample from

a suspect

    • Paternity
mass production of gene products
Mass Production of Gene Products
  • Most are grown in cell culture using bacteria
  • E. coli can host the most plasmids, that is why it is used the most
  • Yeast is often better for manufacture of eukaryotic gene products
  • Study into using whole animals in gene product manufacture is underway
genetically modified organisms gmo s

Agrobacteriumtumefaciens

DNA containinggene for desired trait

Plant cell

1

2

3

Tiplasmid

RecombinantTi plasmid

Insertion ofgene into plasmidusing restrictionenzyme and DNAligase

Introductioninto plantcells inculture

Regenerationof plant

T DNA

T DNA

Plant with new trait

Restriction site

Genetically Modified Organisms (GMO’s)
  • New genetic varieties of animals and plants are being produced
    • A plant with a new trait can be created using the Ti plasmid
gmo s
GMO’s
  • “Golden rice” has been genetically modified to contain beta-carotene
  • This rice could help prevent vitamin A deficiency
gmo s and the environment
GMO’s and the Environment
  • Genetic engineering involves some risks
    • Possible ecological damage from pollen transfer between GM and wild crops
    • Pollen from a transgenic variety of corn that contains a pesticide may stunt or kill monarch caterpillars