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Genetic Engineering

Genetic Engineering: Enzymes for Dicing and Splicing Nucleic Acids

•Restriction endonucleases-enzymes capable of recognizing foreign DNA

and breaking the phosphodiester bonds between adjacent nucleotides on both strands of DNA

-protects bacteria against incompatible DNA of bacteriophages

-allows biotechnologists to cleave DNA at desired sites

-necessary for recombinant DNA technology

-recognize and clip at palindromes

Restriction Endonucleases

•Make staggered symmetrical cuts that leave short tails called “sticky ends”

-cut four to five bases on the 3’ strand and on the 5’ strand, leaving overhangs on each end

-adhesive tails will base-pair with complementary tails on other DNA fragments or plasmids

Restriction Endonucleases

-restriction fragments: pieces of DNA

produced by restriction endonucleases

-restriction fragment length polymorphisms (RFLPs): differences in the cutting patterns of specific restriction endonucleases

Analysis of DNA

•Gel electrophoresis-produces a readable pattern of DNA fragments

-samples are placed in compartments in a soft agar gel and subjected to an electrical current

-phosphate groups have a negative charge, which causes DNA to move toward the positive pole in the gel

-larger fragments migrate more slowly; smaller fragments migrate more quickly

-position of fragments are determined by staining the gel

-creates a genetic fingerprint

Revealing the Patterns with ElectrophoresisCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

1 2 3 4 5

KnownDNA sizemarkers

Smaller

Larger

Wells

Samples

(b)

© Kathy Park Talaro

Restriction endonucleasesselectively cleave sitesof DNA

1 2 3 4 5

DNA for sample 3

DNA migrates towardpositive electrode.

Size markers

Electrophoresis

Restrictionfragments

Wells

Samples

(+)

(a)

(–)

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Polymerase Chain Reaction: A Molecular Xerox Machine for DNA

•Rapidly increases the amount of DNA in a sample without the need for making cultures or carrying out complex purification techniques

•Sensitive enough to detect cancer from a single cell or diagnose an infection from a single gene copy

•Rapid enough to replicate target DNA from a few copies to billions of copies in a few hours

Polymerase Chain Reaction

•Primers: DNA strands 15 – 30 bases long that serve as landmarks where DNA amplification should begin

•DNA polymerase from thermophilic bacteria

-“Taq” polymerase isolated from Thermus aquaticus

-remain active at elevated temperatures used in PCR

•Thermal cycler: automatically performs the cyclic temperature changes required for PCR

Recombinant DNA Technology

•Remove genetic material from one organism and combine it with that of a different organism

•Bacteria can be genetically engineered to mass produce substances such as hormones, enzymes, and vaccines difficult to synthesize by usual industrial methods

Recombinant DNA Technology•Genetic clones/cloning

-involves removal of a selected gene from an animal, plant, or microorganism and propagated in a host microorganism

-gene must be inserted into a vector (usually a plasmid or a virus)

-vector inserts the gene into the cloning host

-cloning host is usually a bacterium or yeast which can translate the gene into the desired protein

Cloning Vectors

•Plasmids -small, well characterized, easy to manipulate

-can be transferred into appropriate cells through transformation

•Bacteriophages: have the natural ability to inject DNA into bacterial hosts through transduction

•Vectors typically contain a gene that confers drug resistance to their cloning host

-cells can be grown on drug containing media

-only those cells that harbor a plasmid will be selected for growth

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Cloning vectors

• Carry a significant piece of the donor DNA (gene of interest)

• Readily accepted DNA by the cloning host

• Contain an origin of replication

• Contain a selective antibiotic resistant gene

• Ex. Plasmids, phages

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Producing Recombinant DNA is easy• Start with a cloning vector (special plasmid) and DNA with your gene

of choice.• Cut the cloning vector and your desired gene out of the parent

chromosome with specific enzymes.

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Producing Recombinant DNA is easy• Mix the vector and the gene together with a ligase enzyme which

“seals” the DNA together.• Use various techniques to insert the vector +gene into a new cell.

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Producing Recombinant DNA is easy• Grow cell on selective or

differential media to find out which cells possess the recombinant plasmid.

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Modified bacteria• Pseudomonas syringae

– Natural bacteria that grow on plants but promote frost crystals– Alteration of the normal frost gene now prevents frost crystals

from forming on plants (applied with crop duster to compete with the natural bacteria in the field)

• Pseudomonas fluorescens– This bacteria was engineered to contain an insecticide gene. The

bacteria is sprayed on fields with crop dusting planes. The bacteria grow on the plants and when the insects start to eat the plant they will also eat some bacteria with the insecticide. The ingestion of insecticide kills the insects.

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Transgenic plants using bacteria

• Agrobacterium tumefaciens– Ti plasmid contains gene of interest, and is

integrated into plant chromosome– Ex. tobacco, garden pea, rice

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Schematic of Agrobacterium tumefaciens transferring and integrating the Ti plasmid into the plant chromosome.

Fig. 10.11 Bioengineering of plants

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Transgenic animals

• Pharmaceutical production• Knockout mouse

– Tailor-made genetic defects• Cystic fibrosis• Gaucher’s disease• Alzheimer’s disease• Sickle-cell anemia• Allow us to research cures to these diseases

Nkx2.2 KO

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Genotypic Diagnostics:DNA Analysis Using Genetic Probes

•Hybridization-used to identify bacterial species by analyzing the sequences of nitrogenousbases in DNA

-probes: small fragments of single-stranded DNA or RNA complementary to the specific DNA sequence of a particular microbe

-unknown test DNA is extracted from cells and bound to blotter paper

-probes are added to the blotter paper and visible signs that probes have been hybridized to the test DNA

DNA Analysis Using Genetic Probes•Probes can be labeled with chemically luminescent materials that can be measured by “light meters,” which avoids the use of radioactivity

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Blotter papercontaining DNAfrom unknown

organism

Additionof probes

ATGGTCTACCAG

ATGGTC

CTGGTA

Color developmenton the paper

Complementary base pairing

No colordevelopmenton the paper

No complementarybase pairing

Nucleic Acid Sequencing and rRNA Analysis

•One of the most viable indicators of evolutionary relatedness and affiliation is the comparison of 16s rRNA sequences

-16s rRNA is part of the 30s subunit of the bacterial ribosome

-16s rRNA is highly conserved across species and evolutionary time

-perfectly suited for bacterial identification and diagnosis of infection

rRNA Analysis•Fluorescent in situ hybridization (FISH)

-rapidly identifies 16s RNA sequences without first culturing the organism

-relies on dyes to emit visible light in response to UV radiation

-turnaround time for identifying suspect pathogens present in blood cultures has been reduced from 24 hours to 90 minutes

Peptide Nucleic Acid FISH Testing for S. aureusCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Image provided by AdvanDx, Inc.

S. aureusPNA probe

Nucleic acid

Peptide

S. aureuson a slide

rRNA inribosome

Gram-positivecocci in clusters

Positive bloodculture tube

Fluorescentmolecule

90 minutes