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
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
Refers to migration of charged electrical species (DNA, RNA, proteins).
Used for separating: proteins, DNA, RNA
Separation by size -> slide
Various resolutions -> slide
Detect complementary sequences -> slide
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
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.
-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:
Sanger – enzymatic method / chain termination
Maxam & Gilbert – chemical method
DNA has to be prepared -> smaller pieces
-> assembly need lots of computer !
Use DNA Polymerase again
Need 4 reaction:
and ddNTPs (stop elongation ! polymerase can’t ad anything -> chain termination)
è ladder of truncated products
Main difference in detecting the products
labeling the reactions:
è Dye-labeled primers, and Dye-labeled terminators.
All 4 rxt mix in one lane
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)