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Recombinant DNA and Genetic Engineering. Chapter 16. Familial Hypercholesterolemia. Gene encodes protein that serves as cell’s LDL receptor Two normal alleles for the gene keep blood level of LDLs low Two mutated alleles lead to abnormally high cholesterol levels & heart disease.

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Familial hypercholesterolemia
Familial Hypercholesterolemia

  • Gene encodes protein that serves as cell’s LDL receptor

  • Two normal alleles for the gene keep blood level of LDLs low

  • Two mutated alleles lead to abnormally high cholesterol levels & heart disease


Example of gene therapy
Example of Gene Therapy

  • Woman with familial hypercholesterolemia

  • Part of her liver was removed

  • Virus used to insert normal gene for LDL receptor into cultured liver cells

  • Modified liver cells placed back in patient


Results of gene therapy
Results of Gene Therapy

  • Modified cells alive in woman’s liver

  • Blood levels of LDLs down 20 percent

  • No evidence of atherosclerosis

  • Cholesterol levels remain high

  • Remains to be seen whether procedure will prolong her life


Genetic changes
Genetic Changes

  • Humans have been changing the genetics of other species for thousands of years

    • Artificial selection of plants and animals

  • Natural processes also at work

    • Mutation, crossing over


Genetic engineering
Genetic Engineering

  • Genes are isolated, modified, and inserted into an organism

  • Made possible by recombinant technology

    • Cut DNA up and recombine pieces

    • Amplify modified pieces


Discovery of restriction enzymes
Discovery of Restriction Enzymes

  • Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack

  • Discovered bacteria have an enzyme that chops up viral DNA


Specificity of cuts
Specificity of Cuts

  • Restriction enzymes cut DNA at a specific sequence

  • Number of cuts made in DNA will depend on number of times the “target” sequence occurs


Making recombinant dna
Making Recombinant DNA

5’

G

A A T T C

3’

C T T A A

G

one DNA fragment

another DNA fragment

5’

G

A A T T C

3’

5’

C T T A A

G

3’

In-text figurePage 254


Making recombinant dna1
Making Recombinant DNA

nick

5’

G

A A T T C

3’

3’

C T T A A

G

5’

nick

DNA ligase action

G

A A T T C

C T T A A

G

In-text figurePage 254


Using plasmids
Using Plasmids

  • Plasmid is small circle of bacterial DNA

  • Foreign DNA can be inserted into plasmid

    • Forms recombinant plasmids

    • Plasmid is a cloning vector

    • Can deliver DNA into another cell


Using plasmids1
Using Plasmids

DNA

fragments

+

enzymes

recombinant

plasmids

host cells containing recombinant plasmids

Figure 16.4Page 255


Making cdna

mRNA transcript

Making cDNA

mRNA–cDNA hybrid

single-stranded cDNA

double-stranded cDNA

Figure 16.5Page 255


Amplifying dna
Amplifying DNA

  • Fragments can be inserted into fast-growing microorganisms

  • Polymerase chain reaction (PCR)


Polymerase chain reaction
Polymerase Chain Reaction

  • Sequence to be copied is heated

  • Primers are added and bind to ends of single strands

  • DNA polymerase uses free nucleotides to create complementary strands

  • Doubles number of copies of DNA


Polymerase chain reaction1

DNA heated to

90°– 94°C

Primers added to

base-pair with

ends

Mixture cooled;

base-pairing of

primers and ends

of DNA strands

DNA polymerases

assemble new

DNA strands

Double-stranded

DNA to copy

Polymerase Chain Reaction

Stepped Art

Figure 16.6Page 256


Polymerase chain reaction2

Mixture cooled; base-pairing between primers and ends of single DNA strands

DNA polymerase action again doubles number of identical DNA fragments

Mixture heated again; makes all DNA fragments unwind

Polymerase Chain Reaction

Stepped Art

Figure 16.6Page 256


Dna fingerprints
DNA Fingerprints single DNA strands

  • Unique array of DNA fragments

  • Inherited from parents in Mendelian fashion

  • Even full siblings can be distinguished from one another by this technique


Tandem repeats
Tandem Repeats single DNA strands

  • Short regions of DNA that differ substantially among people

  • Many sites in genome where tandem repeats occur

  • Each person carries a unique combination of repeat numbers


Rflps
RFLPs single DNA strands

  • Restriction fragment length polymorphisms

  • DNA from areas with tandem repeats is cut with restriction enzymes

  • Because of the variation in the amount of repeated DNA, the restriction fragments vary in size

  • Variation is detected by gel electrophoresis


Gel electrophoresis
Gel Electrophoresis single DNA strands

  • DNA is placed at one end of a gel

  • A current is applied to the gel

  • DNA molecules are negatively charged and move toward positive end of gel

  • Smaller molecules move faster than larger ones


Analyzing dna fingerprints
Analyzing DNA Fingerprints single DNA strands

  • DNA is stained or made visible by use of a radioactive probe

  • Pattern of bands is used to:

    • Identify or rule out criminal suspects

    • Identify bodies

    • Determine paternity


Genome sequencing
Genome Sequencing single DNA strands

  • 1995 - Sequence of bacterium Haemophilus influenzae determined

  • Automated DNA sequencing now main method

  • Draft sequence of entire human genome determined in this way


Nucleotides for sequencing
Nucleotides for Sequencing single DNA strands

  • Standard nucleotides (A, T, C, G)

  • Modified versions of these nucleotides

    • Labeled so they fluoresce

    • Structurally different so that they stop DNA synthesis when they are added to a strand


Reaction mixture
Reaction Mixture single DNA strands

  • Copies of DNA to be sequenced

  • Primer

  • DNA polymerase

  • Standard nucleotides

  • Modified nucleotides


Reactions proceed
Reactions Proceed single DNA strands

  • Nucleotides are assembled to create complementary strands

  • When a modified nucleotide is included, synthesis stops

  • Result is millions of tagged copies of varying length


Recording the sequence

Figure 16.8 single DNA strandsPage 258

T C C A T G G A C C

T C C A T G G A C

Recording the Sequence

T C C A T G G A

T C C A T G G

T C C A T G

T C C A T

T C C A

electrophoresis

gel

T C C

  • DNA is placed on gel

  • Fragments move off gel in size order; pass through laser beam

  • Color each fragment fluoresces is recorded on printout

T C

one of the many fragments of

DNA migrating

through the gel

T

one of the DNA fragments

passing through a laser beam

after moving through the gel

T C C A T G G A C C A


Gene libraries
Gene Libraries single DNA strands

  • Bacteria that contain different cloned DNA fragments

    • Genomic library

    • cDNA library


Using a probe to find a gene
Using a Probe to Find a Gene single DNA strands

  • You want to find which bacteria in a library contain a specific gene

  • Need a probe for that gene

    • A radioisotope-labeled piece of DNA

    • Will base-pair with gene of interest


Use of a probe

Colonies on plate single DNA strands

Use of a Probe

Cells adhere

to filter

Cells are lysed;

DNA sticks

to filter

Probe is

added

Location where probe binds forms

dark spot on film, indicates colony with gene

Figure 16.9Page 259


Engineered proteins
Engineered Proteins single DNA strands

  • Bacteria can be used to grow medically valuable proteins

    • Insulin, interferon, blood-clotting factors

    • Vaccines


Cleaning up the environment
Cleaning Up the Environment single DNA strands

  • Microorganisms normally break down organic wastes and cycle materials

  • Some can be engineered to break down pollutants or to take up larger amounts of harmful materials


The ti plasmid
The Ti plasmid single DNA strands

  • Researchers replace tumor-causing genes with beneficial genes

  • Plasmid transfers these genes to cultured plant cells

plant cell

foreign gene

in plasmid

Figure 16.11Page 261


Engineered plants
Engineered Plants single DNA strands

  • Cotton plants that display resistance to herbicide

  • Aspen plants that produce less lignin and more cellulose

  • Tobacco plants that produce human proteins

  • Mustard plant cells that produce biodegradable plastic


First engineered mammals
First Engineered Mammals single DNA strands

  • Experimenters used mice with hormone deficiency that leads to dwarfism

  • Fertilized mouse eggs were injected with gene for rat growth hormone

  • Gene was integrated into mouse DNA

  • Engineered mice were 1-1/2 times larger than unmodified littermates


Cloning dolly
Cloning Dolly single DNA strands

1997 - A sheep cloned from an adult cell

  • Nucleus from mammary gland cell was inserted into enucleated egg

  • Embryo implanted into surrogate mother

  • Sheep is genetic replica of animal from which mammary cell was taken


Designer cattle
Designer Cattle single DNA strands

  • Genetically identical cattle embryos can be grown in culture

  • Embryos can be genetically modified

    • create resistance to mad cow disease

    • engineer cattle to produce human serum albumin for medical use


The human genome initiative
The Human Genome Initiative single DNA strands

Goal - Map the entire human genome

  • Initially thought by many to be a waste of resources

  • Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes

  • Sequencing was completed ahead of schedule in early 2001


Genomics
Genomics single DNA strands

  • Structural genomics: actual mapping and sequencing of genomes of individuals

  • Comparative genomics: concerned with possible evolutionary relationships of groups of organisms


Using human genes
Using Human Genes single DNA strands

  • Even with gene in hand it is difficult to manipulate it to advantage

  • Viruses usually used to insert genes into cultured human cells but procedure has problems

  • Very difficult to get modified genes to work where they should


Can genetically engineered bacteria escape
Can Genetically Engineered Bacteria “Escape”? single DNA strands

  • Genetically engineered bacteria are designed so that they cannot survive outside lab

  • Genes are included that will be turned on in outside environment, triggering death


Ethical issues
Ethical Issues single DNA strands

  • Who decides what should be “corrected” through genetic engineering?

  • Should animals be modified to provide organs for human transplants?

  • Should humans be cloned?


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