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CHAPTER 20

GENETIC

TECHNOLOGY

Prepared by

Brenda Leady, University of Toledo

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Recombinant DNA technologyUse of laboratory techniques to isolate and

manipulate fragments of DNA Recombinant DNA contains DNA from 2 or

more sources Once inside a host cell, recombinant

molecules are replicated to produce identical copies or clones

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Gene cloning

Want copies of a gene for study or use Obtain lots of gene product- mRNA or

protein

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Step 1 in gene cloning

Vector DNA as a carrier for the DNA segment to be cloned

When a vector is introduced into a living cell, it can replicate making many copies

Common vectors are plasmid or viral Also need the gene of interest from

chromosomal DNA digest

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Step 2

Insert chromosomal DNA into vector Cut DNA using restriction enzymes or restriction

endonucleases Made naturally by bacteria as protection against

bacteriophages Cuts at specific known restriction sites Most restriction sites palindromic May produce sticky ends DNA ligase must be used to permanently link DNA

Result may be Recircularized vector with no gene of interest inserted Recombinant vector with gene of interest inserted

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Vector carries a selectable markerPresence of antibiotics selects for cells

expressing ampR gene – contains plasmidampR gene codes for b-lactamase that

degrades ampicillin, which normally kills bacteria

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Step 3

Goal for recombinant vector to be taken up by bacteria

Transformation – plasmid vector Transfection – viral vector After treatment, only cells with the plasmid

will grow on plates treated with ampicillin

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To eliminate recircularized vectors from further examination, lacZ gene part of vector

Insertion of chromosomal DNA disrupts lacZ gene

lacZ codes for b-galactosidase which cleaves colorless X-Gal into a blue dye

Recircularized plasmids will form blue colonies Recombinant vectors will form white colonies

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DNA library Treatment of chromosomal DNA with restriction

enzymes yields thousands of different fragments DNA library- collection of many recombinant

vectors each with a fragment of chromosomal DNA

2 types of common DNA librariesGenomic – inserts derived from chromosomal DNAcDNA – use reverse transcriptase to make DNA from

mRNA of interest (complementary DNA) - lacks introns so simpler to use

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Use colony hybridization to identify colonies with gene of interestMaster plate with bacterial cells containing

recombinant vector with different pieces of chromosomal DNA

Use single-stranded DNA probeDark spots correspond to colonies with gene

of interest

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Blotting Methods Can Identify Gene Families

Southern blotting can detect the presence of a particular gene within a mixture of many chromosomal fragments separated on a gel

Another common use of Southern blotting is to identify gene families Two or more genes are derived from the same ancestral gene

The members of a gene family are homologous, having similar but not identical DNA sequences Distinguish homologous members of a gene family within a

single species or identify homologous genes among different species

ElectrophoresisSeparates macromolecules based on charge and size

DNA is negative – moves toward positive electrode Smaller fragments move faster/farther than large fragments

Bands can be stained or probed Southern blotting of a gel

Locations where probe binds appear dark on x-ray filmHigh stringency – detects close match between probe

and chromosomal DNALow stringency – detects homologous genes

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Polymerase chain reaction (PCR) Copy DNA without vectors and host cells Goal to make many copies of DNA in a defined region Uses high concentration of two primers that are

complementary to sequences at the ends of the DNA region to be amplified, deoxynucleoside triphosphates (dNTPs), and a heat-stable form of DNA polymerase called Taq polymerase

Sample of DNA taken through repeated cycles of denaturation, annealing and synthesis Thermocycler automates this process

After 30 cycles of amplification, a DNA sample will increase 230-fold

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GenomicsRefers to the molecular analysis of the entire

genome of a species2 types

Structural – direct DNA analysis – mapping Functional – studying expression – which genes

turned on or off in particular cells

ProteomicsUsed to identify and study groups of proteins

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BACs and YACs

Most plasmid and viral vectors accept a few to tens of thousands of nucleotides

Bacterial artificial chromosomes (BAC) can contain up to 500,000 bp

Yeast artificial chromosomes (YAC) can take up to 2 million nucleotides

Used in first phases of mapping – determining relative locations of inserted chromosomal pieces on intact chromosome

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ContigSeries of clones that

contain overlapping pieces of chromosomal DNA

Overlapping regions allow researchers to identify their order along the chromosome

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DNA sequencing Determines base sequence of DNA Dideoxy chain-termination method or dideoxy

sequencing Dideoxynucleoside triphosphates (ddNTPs) are missing the

3’ –OH group and will terminate the chain 4 tubes with many copies of single stranded DNA of interest

Each tube has a different radiolabelled dNTP DNA polymerase will make complementary strand until

dNTP inserted and chain terminates After electrophoresis, DNA sequence can be read by

reading which base is at the end of the DNA strand Procedure has been automated using fluorescent dyes

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Microarray or gene chip Used in functional genomics Monitors expression of thousands of genes

simultaneously Small slide dotted with many different short sequences of

single-stranded DNA from known genes RNA isolated from sample cells used to make

fluorescently labeled cDNA cDNA that is complementary to the DNA in the microarray

will hybridize Measure fluorescence intensity Identifies genes that have been transcribed in a particular

cell under a given set of circumstances

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Proteomics

Entire collection of a species’ proteins Due to gene regulation, any given cell will

produce only a subset of its proteome What proteins are made depends on what

type of cell it is, stage of development, and environmental conditions

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2D gel electrophoresis

Can separate thousands of different proteins in a cell extract

Sample of proteins loaded onto tube shaped gel

Isoelectric focusing – separates proteins according to their net charge at a given pH

Tube gel then placed on slab gel that separates proteins by molecular mass

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Mass spectroscopy Proteins can be identified by their unique amino acid

sequence Tandem mass spectrometry uses 2 spectrometers

First measures mass of a given peptide Second measures mass after the peptide has been digested

into smaller fragments Researcher could determine the possible codon

sequences that could encode such a peptide More than one sequence is possible due to degeneracy of

genetic code Using computer software, entire genomic sequence scanned Can locate match between codon sequence and specific gene

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Biotechnology

Technologies that involve the use of living organisms to benefit humans

Use began about 1200 years ago with livestock domestication

More recently associated with molecular genetics

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Insulin

In 1982, US FDA approved sale of human insulin made by recombinant bacteria

Prior to 1982, insulin isolated from cattle Some people developed allergies and had to use

cadaver insulin Insulin composed of 2 polypeptides – A and B

A and B coding sequence inserted into E.coli Fusion proteins extracted and b-galactosidase

removed Purified A and B chain mixed to form functional protein

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Bioremediation

Use of microorganisms to decrease pollutants in the environment

Enzymes produced by microorganism can alter or transform toxic pollutant structure – biotransformation

May degrade toxic form into less complex, nontoxic metabolites – biodegradation

1980, first patented recombinant microorganism Not a commercial success

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Transgenics An organism that carries genes introduced using molecular

techniques such as gene cloning Genetically modified organisms (GMOs)

Gene replacement – cloned gene recombines with normal gene on a chromosome Only 1 of 2 copies replaced creating hetrozygote Heterozygotes can be crossed to yield homozygotes

Gene knockout – if cloned gene is a mutation that inactivates function, homozygote will not have gene function

Application in studying human disease Used as models to study disease Used to test effect of therapies

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Molecular pharming

Production of medically important proteins in livestock mammary glands

Certain proteins more likely to function when expressed in mammalsPost-translational modificationDegraded or improperly folded in bacteriaHigh yield in cows

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Strategy to clone gene next to the promoter of a gene specifically expressed in mammary cells

Vector injected into oocyte, fertilized and implanted

Milk containing hormone purified for protein

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Transgenic plants Somewhat easier because plant cells are totipotent Cloned genes can be introduced into somatic tissue and entire

plant regenerated with hormonal treatments Agrobacterium tumefaciens naturally infects plant cells and

causes tumors Contains Ti plasmid that integrates into host chromosome Codes for plant growth hormones that form crown gall tumor

Ti plasmid modified to introduce cloned genes KanR used as a selectable marker for kanamycin resistance Contain convenient restriction sites

Transformed cells plated on media with kanamycin (kills nontransformed cells) and carbenicillin (kills Agrobacterium)

Transgenic plants are approved for human consumption

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Cloning mammals

Identical twins are genetic clones from a single fertilized egg that split early in development

Plants can be cloned from somatic cells Believed for decades that mammalian

somatic cells could not be used for cloning 1996, Dolly is the first cloned lamb

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Mammary cells from adult sheep removed Fused diploid mammary cell with enucleated

sheep oocyte Zygote implanted Dolly and donor were almost genetically

identicalSame set of genesMinor differences due to differences in

mitochondrial DNA and maternal effect genes Achieved in several mammal species

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DNA fingerprinting/ DNA profiling

Identifies particular individual using properties of their DNA

Chromosomal DNA produces series of bands on a gel Unique pattern of bands used Automated using PCR to amplify short tandem repeat

sequences (STRs) Uses

Identify different species Forensics – 1986 first use in US court system Paternity testing

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Blaese and Colleagues Performed the First Gene Therapy to Treat ADA Deficiency

Gene therapy introduces cloned genes into living cells to cure disease

More than 4,000 diseases involve a single gene abnormality

Adenosine deaminase deficiency prevents proper metabolism of nucleosides

Deoxyadenosine accumulates and results in destruction an B and T cells

Leads to severe combined immunodeficiency syndrome (SCID) Fatal at early age (1 or 2 years old)

3 treatment approachesBone marrow transplantPurified ADA enzymeGene therapy

Gene therapySeptember 14, 1990Remove lymphocytes from girlTreat with retroviral vector containing ADA geneReturn cells to her bloodstream

Results suggest that this first gene therapy trial may offer benefit but patients also treated with ADA