Manipulating DNA:tools and techniques

Chapter 12

Tools for gene manipulation

Action ‘Tool‘Cut DNA into fragments at precise locations

Restriction enzymes (p. 422-423)

Separate fragments by size Electrophoresis (p. 423-424)

Find particular DNA fragments

Probes (p. 424-425)

Join DNA fragments Ligase enzyme (p. 425-426)

Transport DNA into cells Vectors (p. 426)

Obtain multiple copies of gene

Gene cloning (p. 428)

TOOLS: Things used by Genetic Engineers to manipulate DNA

Resources

• Try McGraw Hill Site(http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#)

• Use the DNA interactive website to review the processes that we will use at Melbourne Uni.http://www.dnai.org/b/index.html

We will be interested in the information found under the tab/title of "Techniques".- cutting & pasting- sorting & sequencing- amplifying (PCR)

Making recombinant DNAHow...Restriction Enzymes = scissors

specific, man made & naturally occurring

Use plasmids (circular DNA found in Bacteria) - often use ones that are resistant to particular antibioticsE.coli is often used: well understood, fast replication

Why...For human use: for artificial synthesis of an natural substance (eg: insulin)

Restriction Enzymes = cuttingOccur naturally in microbes (eg: bacteria). Can produce blunt or sticky endsDNA is dissolved & a restriction enzyme is addedDNA will be cut into 2 or more pieces (provided recognition site is present)Length of fragment varies with location of recognition site

Naming (restriction) enzymes

• Restriction enzymes are names after the bacterium from which they are isolatedRestriction Enzyme Bacterium

EcoRI Escherichi coliStrain RY131st endonuclease isolated (I)

BamHI Bacillus amyloliquefaciensStrain H1st endonuclease isolated (I)

HindIII Haemophilus influenzaeStrain Rd3rd endonuclease isolated (III)

ProbesProbes = locate targets

Can be a single stranded DNA fragment or an RNA fragment

DNA is denatured to separate the strands. the Probe will locate the target (region of a single strand)

Fluorescence / radio-active markers are usually added to a probe for easy recognitionFISH = fluorescence in situ hybridisation

ElectrophoresisElectrophoresis = sorts out (cut)

fragments

Smaller fragments move faster (piggy-back analogy)

Distance moved by and unknown band (fragment) is compared against known sizes to work out how big it is

It works because DNA has a net negative charge (because of phosphate group)

So DNA is put in wells at the negative end and is attracted / moves towards the positive end.

Ligase

Ligase = joins double-stranded DNA

The enzyme that catalyses te joining of DNA as the sugar-phosphate backbone

Thus producing longer DNA or circular DNA (if there are complementary sticky ends)

Polymerase Chain Reaction (PCR)• PCR enables researchers to produce millions of copies of a specific DNA sequence in

approximately two hours• This process does not require the need to use bacteria for amplifying DNA.• PCR depends on the enzyme DNA polymerase to amplify a sample of DNA. This enzyme

comes from a bacterium that lives in hot springs and is known as the Taq Polymerase

• STEP 1: DENATURINGTarget DNA samples are heated to 95°C so that the target DNA dissociates into single strands.

• STEP 2: BIND PRIMERSNext the sample is cooled to between 50 and 65°C which allows the left and right primers to anneal (bonded) to their complementary sequences. The primers are designed to bracket the DNA region of interest.

• STEP 3: EXTEND PRIMERSTemperature is raised to 72°C which allows the Taq Polymerase to attach at each priming site and synthesise a new DNA strand. A supply of free nucleotides is needed for this step as two new complete double strands are formed.

These steps are repeated until we get a DNA sequence with the desired base sequence, which normally occurs after the 3rd cycle.

VectorsVectors (eg: plasmids)The plasmid is cut using a restriction

enzyme that creates sticky ends.

DNA passenger fragments are prepared using the same restriction enzyme.

The passenger DNA fragments & plasmids are mixed with the enzyme ligase.

Plasmids are reintroduced to bacterial cells using an electrical pulse or heat shock.