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Chapter 23 (Part 1). Recombinant DNA Technology. Recombinant DNA Technology. Methods for isolating, manipulating, and amplifying identifiable DNA sequences. Allows us to study the structure and function of individual genes.

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Chapter 23 (Part 1)

Recombinant DNA Technology

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Recombinant DNA Technology

  • Methods for isolating, manipulating, and amplifying identifiable DNA sequences.

  • Allows us to study the structure and function of individual genes.

  • Allows for the directed genetic manipulation of organism (modify gene function, insert novel genes)

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  • Clone: a collection of molecules or cells, all identical to an original molecule or cell

  • To "clone a gene" is to make many copies of it - for example, in a population of bacteria

  • Gene can be an exact copy of a natural gene

  • Gene can be an altered version of a natural gene

  • Recombinant DNA technology makes it possible

  • Allows for in vitro manipulation of a individual gene

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Tools Needed for Cloning(Think of it as a cutting and pasting process)

  • cDNA or genomic library (source of DNA to cut)

  • Plasmid (where you want to paste it)

  • Restriction enzymes (scissors)

  • DNA ligase (paste)

  • E. coli (biological machine needed to amplify DNA)

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  • Naturally occurring extrachromosomal DNA

  • Self replicating circular double stranded DNA molecules that have their own origin of replication

  • Usually present in multiple copies per cell

  • Plasmids can be cleaved by restriction enzymes, leaving sticky ends

  • Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid

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Cloning Vector

Required features

  • Origin of replication

  • Selectable marker

  • Screenable marker for recombinant molecules

  • Cloning sites

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Restriction Enzymes

  • Bacteria protect themselves from attack by viruses and other bacteria using a restriction/modification system.

  • Allows bacteria to recognize and destroy foreign DNA

  • Bacteria contain DNA methylases that modify their chromosomal DNA at specific sequences.

  • Also contain restriction endonucleases that recognize and cleave these same sequences when they are not methylated

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DNA methylase

DNA methylase





Restriction endonuclease

Restriction endonuclease





Restriction Modification System

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Restriction Enzymes

  • Type I – Contain methylase and endonulcease fuctions. Require ATP for hydrolysis and S-adenosylmethionine for methylation

  • Type II – contain only endonulcease function,. Does not require ATP for hydrolysis.

  • Both types recognize palindrome sequences (sequences that read the same if read forward or backwards – e.g. “BOB” or “DEED”

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Type II Restriction Enzymes

  • Names use 3-letter italicized code:

  • 1st letter - genus; 2nd,3rd - species

  • Following letter denotes strain

  • EcoRI is the first restriction enzyme found in the R strain of E. coli

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Sticky-end cutter

Blunt-end cutter

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Restriction Enzymes

  • Restriction enzymes can recognize specific 4 base, 6 base, 8 base sequences.

  • The probability that a given piece of DNA will contain a specific restriction site is = n4

  • n = the number of bases in the restriction site

  • So for a 6 base cutter (64), you would expect to find your site every ~1300 base pairs. So in a 10,000 bp fragment there is likely to by 7 or 8 restriction sites corresponding to your enzyme.

  • You can characterize DNA fragments using gel electrophoresis

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T4 DNA Ligase

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  • All of the previous steps were performed in vitro.

  • We have generated a very small amount of a recombinant plasmid

  • Need to amplify in bacteria to get enough to work with.

  • Transformation – process to mobilize DNA into bacterial host

  • Select for transformed bacteria on specific antibiotic that corresponds to the antibiotic resistance gene present on the plasmid

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How to produce a recombinant protein

0.1 to 1% of cellular protein

10 to 70% of cellular protein

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Cloning a gene from a DNA libraries

  • Any particular gene may represent a tiny, tiny fraction of the DNA in a given cell

  • Can't isolate it directly

  • Trick is to find the fragment or fragments in the library that contains the desired gene

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Library Screening

  • DNA probe hydridization

  • Requires that you know the protein or amino acid sequence of the gene of interest.

  • Need to denature (make single stranded) and immobilize the DNA from each clone of the library to a filter (nitrocellulose or nylon)

  • Make a labeled single stranded DNA/RNA probe (can use radioactive of fluorescent analogous of specific nucleotide triphosphates)

  • Labeled single stranded DNA/RNA fragments will base pair (hydridize) with the target DNA on the filter

  • Identify clones that are labeled.

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DNA hydridization screening for specific gene

  • Requires that you know something about the gene sequence

  • Can get sequence information form purified protein

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Now that we have the gene, what do we do with it?

  • We could use it make a lot of protein in a microbial protein expression system

  • We could use it to genetically manipulate organisms

  • We could use it as a diagnostic tool

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Why use recombinant Proteins?

  • Proteins are often only available in small amounts in a given tissue

  • Tissue sources may not be readily available

  • It is time consuming and expensive to purify protein from tissues

  • It is difficult to obtain absolutely pure protein

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  • Was first purified from human pancreas from cadavers and then from pig pancreas.

  • Genentec expressed insulin gene in microbial host

  • Can grow microbes in large fermenters to produce unlimited supply of insulin.

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Product name 

Protein type



Adagen (Adenosine deaminase )

An enzyme

Severe combined immunodeficiency disease (SCID)


Genotropin (Recombinant growth hormone)

A hormone

Growth hormone deficiency (GHD) in children

Pharmacia & Upjohn

Humalog (Recombinant human insulin)

A hormone


Eli Lilly

Nabi-HB (Anti-Hepatitis B)

 An antibody



Novo Seven (Recombinant coagulation factor VIIa)

A modified factor

Hemophillia patients with inhibitors

Novo Nordisk

Ontak (Diphtheria toxin-interleukin-2)

A fusion protein

Cutaneous T-cell lymphoma (CTCL)

Ligand Pharmaceuticals

Roferon-A (Recombinant interferon alfa-2a)

A modifier

Hairy cell leukemia or AIDS-related Kaposi's sarcoma

Hoffmann-La Roche

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Recombinant proteins are also important to research

  • For enzyme analysis need pure protein

  • For structural analysis need lots (milligram amounts) of very pure protein

  • Need pure proteins to make diagnostic tools such as antibodies

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Genetic Modification of Higher Organisms

  • Can introduce gene into animals and plants

  • These modified organism are powerful research tools to study the effect of a specific gene product on metabolism, development etc….

  • Has also been used to develop improved agricultural products

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Plant Genetic Engineering

Improved Agricultural Production

  • Herbicide Resistance

  • Pest Resistance

    Improved Nutrition

  • Vitamins - Golden Rice, Vitamin E

  • Increase essential Amino Acid Content

    Chemical Synthesis

  • Bio-plastics

  • Bio-diesel

  • Lubricants/detergents

  • Rubber

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GMO Concerns

  • Ecological Concern

  • Potential Food Allergens

  • Antibiotic Resistance

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GMO Benefits

  • Lower application of herbicides and pesticides

  • Creation of foods with increased nutrition

  • Creation of bio-based alternative to petroleum based products