DNA CLONING - A Quick Overview

• lOther names: “Recombinant DNA Technology” or “Genetic Engineering”

• lIdea – pick up a “desirable” characteristic and copy / clone it

• lExtreme version? – clone whole organism (ie. Dolly the sheep)

• lTypically focus on one characteristic / set of genes at a time

 

Example products of Gene Cloning:

• •insulin for diabetics
• •factor VIII for males suffering from hemophilia A
• •factor IX for hemophilia B
• •human growth hormone (GH)
• •erythropoietin (EPO) for treating anemia
• •three types of interferons
• •several interleukins
• •granulocyte-macrophage colony-stimulating factor (GM-CSFstimulating bone marrow
• •tissue plasminogen activator (TPA) dissolving blood clots
• •adenosine deaminase (ADA) for treating immunodeficiency 
• •angiostatin and endostatin for trials as anti-cancer drugs
• •parathyroid hormone 

 

So you want to tinker with life itself?   5 General steps to clone a DNA sequence:

1. 1.Cut DNA at precise locations (restriction endonucleases chop specific sites)
2. 2.Join 2 fragments together (enzyme DNA ligase joins DNA pieces) to get “recombinant DNA”  (composite molecules of DNA containing covalently-linked segments.
3. 3.Incorporate DNA into “cloning vectors” such as plasmids (small DNA rings) or viral DNA
4. 4.Transfer DNA (from test tube / cloning vector) into host cell to copy / reproduce the DNA or amplify via PCR
5. 5.Have a method to identify / isolate “host cells” containing newly-made “recombinant DNA”  

 

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RESTRICTION MODIFICATION - Enzymatic cutting of DNA / RNA

Since we cannot cut individual molecules of DNA ourselves, it has found that using "restriction endonucleases" (enzymes) which are found naturally, provides a remarkable degree of accuracy for cutting and rejoining DNA / RNA fragments.

Key terms:

• Bacteriophage: a virus capable of replicating within a bacterial cell
• •Restriction: the chopping / fragmenting of DNA
• •Modification: change / modify infecting DNA to make it resistant to restriction. Usually accomplished by methylating (+ CH3) or glycosylating (+ sugar) specific bases at specific sites

Example Enzymatic Restriction:

http://alpha.furman.edu/~lthompso/bgy30/dnatech/ttVectors.html 

 

 

Example Restriction in Nature:  Virus / Bacteriophage T4

• lBacteriophage T4 has no cytosine (has HMC / hydroxymethylcytosine)
• lEarly in viral infection, the T4 DNA codes for two endonucleases which chop the host DNA into fragments (at cytosine site)
• lFragments containing cytosine are then modified by T4 enzymes to stop further restriction.
• lGenerally – virus enters cell, takes over genetic machinery and makes copies of itself (at the expense of the host)

Example Restriction:  ECO RI 

• 1.ECO R1 (a restriction endonuclease from E.coli) cleaves DNA with specific sequence:  5’…GAATTC…3’
• 2.Modification enzyme ECO R1 Methylase  methylates one Adenine (A) on each strand within the ECO R1  (palindromic) sequence:
•               5’…GAATTC…3’
•               3’…CTTAAG…5’

    This "restriction" then blocks further restriction / chopping

   

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CLASSES OF RESTRICTION ENDONUCLEASES

Specificity of Restriction Endonucleases

• lR.E. are found in a wide range of bacterial species
• lBiological function? To recognize and cleave / destroy foreign DNA such as infecting viruses
• lA cell’s own DNA is protected by having the recognition sequences methylated
• l
• l3 General Types of R.E.
• Class I,  Class II, and Class III

 

Classes of Restriction Endonucleases

Class I and III

• lTend to be large, multi-subunit complexes
• lHave both endonuclease and methylase activities
• lType I – cleave DNA at random sites up to 1000 bp (base pairs) from recognition sequence
• lType III – cleave DNA at ~25bp from recognition site
• lBoth types – require energy (in form of ATP)

Class II

• lTend to  be much simpler
• lCleave DNA at / within recognition sequence
• lRecognize short, specific palindromes
• lForms either “Blunt” (strands cut across from each other) or “Cohesive” ends (cuts DNA at a point several bp away from complementary bases

 

Class II RE – Cohesive (“Sticky”) Ends 

http://www.escience.ws/b572/L5/L5.htm

Class II RE – Blunt Ends

http://www.escience.ws/b572/L5/L5.htm

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Enzymes used in Recombinant DNA Technology:

1. lType II RE – cleave DNA at specific sequences
2. lDNA Ligase – joins two fragments
3. lDNA Polymerase – fills gaps by adding nucleotides
4. lReverse Transcriptase – make DNA copy of RNA
5. lPolynucleotide Kinase – add phosphate to 5’ end
6. lTerminal Transferase – add “tails” to 3’-OH end
7. lExonuclease III - remove nucleotides fr5om 3’ end
8. lBacteriophage lambda – remove nucleotides from 5’ end to expose singled-stranded 3’ends
9. lAlkaline Phosphatase – removes terminal Pi from 5’3’ ends

 

Specific Class II R.E.

1. lECO RI (E.coli RY13) - cohesive ends
2. lBAM H1 (B.amyloliquifaciens) - cohesive
3. lHIND III (H.influenzae Rd) - cohesive
4. lSma I (Serratia marcesens) - blunt ends
5. lHIND II (H. influenzae Rd) - blunt ends
6. lHae II (H.aegyptius) - cohesive

    

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