Restriction

Restriction Enzymes

Cleave DNA at specific sites. ->slide

Recognition motifs – short DNA sequence motif

Note: not every combination has an enzyme that recognizes it.

Sticky or blunt ends

Restriction mapping

A restriction map is a type of physical map

Restriction sites also serve as molecular markes

1.Example: neurodegenerative disorder Huntington’s chorea

Patient has 5kb HindIII fragment

Parents have 4.8 kb fragment

è 200 new bases inserted during transmission

è Huntington gene is prone to this insertion mutation

Electrophoresis

Refers to migration of charged electrical species (DNA, RNA, proteins).

Used for separating: proteins, DNA, RNA

Separation by size -> slide

Various resolutions -> slide

Gel-transfer hybridization

Detect complementary sequences -> slide

Hybridization:

Southern Blot (DNA and DNA probe)

Northern Blot (RNA with DNA or RNA probe)

Western Blot (Protein and Antibody)

PCR polymerase chain reaction

Purpose: make huge number of copy of a gene

(for sequencing or cloning)

Works with DNA polymerase

Need:

• Template (eg genomic DNA)

• Primer: All DNA polymerases need a primer. Need an existing piece of nucleic acid where it can ADD something to its 3’ end. (order from company)
• dNTPs (A,G,T,C)

3 major steps in a PCR, which are repeated for 30 or 40 cycles (exponentially amplify). This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

1. Denaturation at 94°C :

Double strand melts open to single stranded DNA,

all enzymatic reactions stop (for example : the extension from a previous cycle).

2. Annealing depends on primers used

The primers are jiggling around, caused by the Brownian motion. Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the hydrogen bond is so strong between the template and the primer, that it does not break anymore.

3. Extension at 72°C :

Ideal working temperature for the polymerase.

Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment.

Cloning

-Storage, amplification, expression

-chromosomal (genomic)/PCR products

-copies of transcrips

Yeast, E.coli = factory to replicate DNA

Important tool: vector = engineered plasmid

Plasmid: extra chromosomal DNA

Naturally occurring (antibiotic resistence)

Replicates independent of Chromosome

100 of copies in cell

Usefull for propagating foreign genes

Specifically designed properties:

• Selectable marjer (antibiotic resistence)
• Cloning site (restriction enzyme recognition)

Sequencing

2 methods

Sanger – enzymatic method / chain termination

Maxam & Gilbert – chemical method

DNA has to be prepared -> smaller pieces

Shotgun

• DNA randomly sheared into small pieces (usually about 1 kb) and subcloned into a "universal" cloning vector.
• Library of subfragments is sampled at random, and a number of sequence reads generated

-> assembly need lots of computer !

Principle Sanger

Use DNA Polymerase again

Need 4 reaction:

template

Primer

dNTPs

and ddNTPs (stop elongation ! polymerase can’t ad anything -> chain termination)

è ladder of truncated products

Automated Sequencing

Main difference in detecting the products

labeling the reactions:

è Dye-labeled primers, and Dye-labeled terminators.

All 4 rxt mix in one lane

Automated readout

All existent Automated DNA Sequencing methods use the Sanger type synthesis

Capillary array electrophoresis DNA sequencing

It is possible to perform 10-12 runs per day, with a daily throughput of about 750,000

bases sequenced per machine.

Separation by charge-to-mass ratio

Site-directed Mutagensis (Michael Smith)

• Specific alteration of residues
• Also unspecific