dna enzyme reactions ligation restriction enzymes helicase enzymes polymerization strand-displacing...

DNA Enzyme reactions

• Ligation• Restriction enzymes• Helicase enzymes• Polymerization• Strand-displacing polymerases

Big Picture - Enzymes• Exonucleases• Endonucleases• Restriction Endonucleases– Type I – cut elsewhere of recognition sites– Type II – cut within recognition sites

http://www.eplantscience.com/index_files/biotechnology/Genes%20&%20Genetic%20Engineering/Tools%20of%20Genetic%20Engineering/biotech_enzymes.php

Ligation

Self-assembled DNA Nanostructures and DNA Devices, Nanofabrication Handbook, Taylor and Francis 2012, with Nikhil Gopalkrishnan, Thom LaBean and John Reif http://www.bio.miami.edu/dana/pix/phosphodiester.jpg

Ligase – “to bind” or “to glue together”

T4 DNA Ligase – a single polypeptide, MW ~ 86,000 daltons, {pH 7.5 – 8.0, 10 mM Mg2+, DTT, NaCl 200 mM to stop reaction}

Restriction Enzymes

Self-assembled DNA Nanostructures and DNA Devices, Nanofabrication Handbook, Taylor and Francis 2012, with Nikhil Gopalkrishnan, Thom LaBean and John Reif

Restriction Endonucleases

• Nicking Enzymes• Restriction Enzymes

Restriction Enzymes

http://www.scq.ubc.ca/restriction-endonucleases-molecular-scissors-for-specifically-cutting-dna/

DNA 101: Enzyme Ligation, Restriction

Sticky ends

DNA ligase

DNA restriction enzyme

www.cs.duke.edu/~reif/paper/peng/.../talk.WalkerDesign.ppt

DNA 101: Enzyme Ligation, Restriction

Sticky ends

DNA ligase

DNA restriction enzyme

www.cs.duke.edu/~reif/paper/peng/.../talk.WalkerDesign.ppthttp://www.angelfire.com/sc3/toxchick/molbiolab/molbiolab01.html

DNA 101: Enzyme Ligation, Restriction

Sticky ends

DNA ligase

DNA restriction enzyme

www.cs.duke.edu/~reif/paper/peng/.../talk.WalkerDesign.ppthttp://www.angelfire.com/sc3/toxchick/molbiolab/molbiolab01.html

Helicase Enzymes • Helicase enzymes are motor proteins that moving along a DNA double

helix to denature its structure (unwind the double helix) independent of temperature.

• In particularly, helicase enzymes directionally break hydrogen bonds between base pairing in DNA double helix.

http://click4biology.info/c4b/3/chem3.4.htmhttp://www.pdbj.org/eprots/index_en.cgi?PDB%3A3BEP

Polymerization

• Denaturation of target (template)– Usually 95oC

• Annealing of primers– Temperature of annealing is dependent on the

G+C content– May be high (no mismatch allowed) or low (allows

some mismatch) stringency• Extension (synthesis) of new strand

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

5’3’

3’5’

Target

1. Denature

2. Anneal primers

3. Extend primers

Two copies of target

1. Denature

2. Anneal primers3. Extend primers

Four copiesof target

AMPLIFICATION BY PCR

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

PCR: First 4 Cycles

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

PCR: Completed Amplification Cycle

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

POLYMERASE CHAIN REACTION

• Primers (may be specific or random)• Thermostable polymerase

– Taq pol– Pfu pol– Vent pol

• Target nucleic acid (template)– Usually DNA– Can be RNA if an extra step is added

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Features of Primers

• Types of primers– Random– Specific

• Primer length– Annealing temperature– Specificity

• Nucleotide composition

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

PCR Primers

• Primers are single-stranded 18–30 b DNA fragments complementary to sequences flanking the region to be amplified.• Primers determine the specificity of the PCR

reaction.• The distance between the primer binding sites

will determine the size of the PCR product.

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Tm

• For short (14–20 bp) oligomers:– Tm = 4° (GC) + 2° (AT)

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

ASSUMPTIONS

• Product produced is product desired– There is always the possibility of mismatch and

production of artifacts– However, if it is the right size, its probably the

right product• Product is from the orthologous locus– Multigene families and pseudogenes

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Thermostable DNA Polymerase: Yellowstone National Park

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Alvin Submersible for Exploration of Deep Sea Vents

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Thermostable Polymerases

Polymerase T ½, 95oC

Extension Rate (nt/sec)

Type of ends

Source

Taq pol 40 min 75 3’A T. aquaticus

Amplitaq (Stoffel

fragment)

80 min >50 3’A T. aquaticus

Vent* 400 min >80 95% blunt

Thermococcus litoralis

Deep Vent* 1380 min ? 95% blunt

Pyrococcus GB-D

Pfu >120 min 60 Blunt Pyrococcus furiosus

Tth* (RT activity)

20 min >33 3’A T. thermophilus

*Have proof-reading functions and can generate products over 30 kbp Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Performing PCR

• Assemble a reaction mix containing all components necessary for DNA synthesis.• Subject the reaction mix to an amplification

program.• Analyze the product of the PCR reaction (the

amplicon).

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

A Standard PCR Reaction Mix

0.25 mM each primer0.2 mM each dATP, dCTP, dGTP, dTTP50 mM KCl10 mM Tris, pH 8.41.5 mM MgCl2

2.5 units polymerase102 - 105 copies of template50 ml reaction volume

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

PCR Cycle: Temperatures

• Denaturation temperature– Reduce double stranded molecules to single stranded molecules– 90–96oC, 20 seconds

• Annealing temperature– Controls specificity of hybridization– 40–68oC, 20 seconds

• Extension temperature– Optimized for individual polymerases– 70–75oC, 30 seconds

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Combinations Of Cycle Temperatures

TEMP FOR COMMENTS94-60-72 Perfect, long

primersHigher temp can be used;maximum annealling temp

94-55-72 Good or perfectlymatched primersbetween 19-24 nt

Standard conditions

94-50-72 Adequate primers Allows 1-3 mismatches/20 nt

94-48-68 Poorly matchedprimers

Allows 4-5 mismatches/20 nt

94-45-65 Unknown match,likely poor

Primers of questionablequality, long-shot PCR

94-37-65 Hail Mary Uncontrolled results

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Thermostable Polymerases

• Taq: Thermus aquaticus (most commonly used)– Sequenase: T. aquaticus YT-1– Restorase (Taq + repair enzyme)

• Tfl: T. flavus• Tth: T. thermophilus HB-8• Tli: Thermococcus litoralis• Carboysothermus hydrenoformans (RT-PCR)• P. kodakaraensis (Thermococcus) (rapid synthesis)• Pfu: Pyrococcus furiosus (fidelity)– Fused to DNA binding protein for processivity

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Amplification Reaction

• Amplification takes place as the reaction mix is subjected to an amplification program.

• The amplification program consists of a series of 20–50 PCR cycles.

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Automation of PCR

• PCR requires repeated temperature changes.• The thermal cycler changes temperatures in a

block or chamber holding the samples.• Thermostable polymerases are used to

withstand the repeated high denaturation temperatures.

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Avoiding Misprimes

• Use proper annealing temperature.• Design primers carefully.• Adjust monovalent cation concentration.• Use hot-start: prepare reaction mixes on ice, place in

preheated cycler or use a sequestered enzyme that requires an initial heat activation.– Platinum Taq– AmpliTaq Gold– HotStarTaq

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Primer Design

• http://biotools.umassmed.edu/bioapps/primer3_www.cgi

• http://arbl.cvmbs.colostate.edu/molkit/rtranslate/index.html

• Avoid inter-strand homologies• Avoid intra-strand homologies• Tm of forward primer = Tm of reverse primer• G/C content of 20–80%; avoid longer than GGGG • Product size (100–700 bp)• Target specificity

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Product Cleanup

• Gel elution– Removes all reaction components as well as

misprimes and primer dimers• Solid phase isolation of PCR product (e.g., spin

columns)• DNA precipitation

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Contamination Control

• Any molecule of DNA containing the intended target sequence is a potential source of contamination.

• The most dangerous contaminant is PCR product from a previous reaction.

• Laboratories are designed to prevent exposure of pre-PCR reagents and materials to post-PCR contaminants.

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Contamination of PCR Reactions

• Most common cause is carelessness and bad technique.• Separate pre- and post-PCR facilities.• Dedicated pipettes and reagents.• Change gloves.• Aerosol barrier pipette tips.• Meticulous technique• 10% bleach, acid baths, UV light• Dilute extracted DNA.

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Strand Displacement Polymerases

Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

The Amplification Phase Step 1

• The exponential amplification process begins with altered targets (single-stranded partial DNA strands with restricted enzyme recognition sites) from the target generation phase.

Lisa Smith & Apollo Kacsinta, Cal State LA, Strand Displacement Amplification

Step 2

• An amplification primer binds to each strand at its complimentary DNA sequence.

Lisa Smith & Apollo Kacsinta, Cal State LA

Step 3

• DNA polymerase uses the primer to identify a location to extend the primer from its 3' end, using the altered target as a template for adding individual nucleotides.

Lisa Smith & Apollo Kacsinta, Cal State LA

Step 4

• The extended primer forms a double-stranded DNA segment containing a complete restriction enzyme recognition site at each end.

Lisa Smith & Apollo Kacsinta, Cal State LA

Step 5

• The restriction enzyme binds to the double stranded DNA segment at its recognition site.

Lisa Smith & Apollo Kacsinta, Cal State LA

Step 6

• The restriction enzyme dissociates from the recognition site after having cleaved only one strand of the double-sided segment, forming a nick.

Lisa Smith & Apollo Kacsinta, Cal State LA

Step 7

• DNA polymerase recognizes the nick and extends the strand from the site, displacing the previously created strand.

Lisa Smith & Apollo Kacsinta, Cal State LA

Step 8

• The recognition site is repeatedly nicked and restored by the restriction enzyme and DNA polymerase with continuous displacement of DNA strands containing the target segment.

Lisa Smith & Apollo Kacsinta, Cal State LA

Step 9

• Each displaced strand is then available to anneal with amplification primers similar to the action in step 2. The process continues with repeated nicking, extension and displacement of new DNA strands, resulting in exponential amplification of the original DNA target.

Lisa Smith & Apollo Kacsinta, Cal State LA

Strand Displacement Amplification

Lisa Smith & Apollo Kacsinta, Cal State LA

Further Reading

• DNAzymes for sensing, nanobiotechnology and logic gate applications – Willner et. al.

• Functional DNA nanotechnology: emerging applications of DNAzymes and aptamers – Liu et. al.

• DNAzymes: From Creation In Vitro to Application In Vivo – Li et. al.

• FRET Study of a Trifluorophore-Labeled DNAzyme – Lu et. al.