CPSC536A - Notes for Class 3



1. Mutations (cont.)
2. Tree of Life
3. Techniques in Molecular Biology
4. Topics in Bioinformatics

1. Mutations (cont.)

In addition to the class of mutations mentioned in section 8 of last week's notes, the following 2 classes exist.

1.2 Insertions and Deletions

Insertion mutations introduce large sequences of DNA into the genome, while Deletion mutations remove large quantities. If the number of base pairs added/removed is not a multiple of 3, this mutation is (in addition) a frame shift mutation. Retro viruses can cause insertions.
(see slide #1)

1.3 Crossover

Crossover mutations occur in a diploid organism (ie. 2 sets of homologous chromosomes) when a DNA part of the first chromosome is built into the second chromosome and vice versa. One DNA of the first chromosome continues on the second chromosome and replaces one DNA there. This DNA crosses over to the other DNA of the first chromosome and completes it. The recombination can lead to huge sequence changes in the genome.
(see slide #2)

2. Tree of Life

The Tree of Life (Phylogenetic Tree) charts a trace-back for all organisms to either the ancestral prokaryote (for bacteria and similar) or the ancestral eukaryote (for higher organisms) (see slide #3). For more on prokaryotes vs. eukaryotes read section 4 of last week's notes. Mitochondria and chloroplasts may have developed as symbionts in prokaryotic organisms (endosymbiont hypothesis). (see slide #4) The internal nodes of the tree are hypothetical and do not necessarily correspond to actual organisms. They represent common differences in the DNA along a path in the tree. The early development (i.e. close to the root) along this tree may be due to simple mutations, while later development involves more recombination mutations (insert & delete, crossover).

3. Techniques in Molecular Biology

3.1 Restriction

The Restriction technique uses Restriction Enzymes to cut DNA at a particular recognition sequence (of only a few base pairs). Not every possible short sequence has a corresponding Restriction Enzyme. Some Restriction Enzymes create blunt ends (eg. Hpa I), others create sticky or staggered ends (eg. Eco RI) (see slide#7). Notice that Mbo II does not cut the DNA at its recognition sequence. (see slide #8)

In nature, Restriction Enzymes play an important role in chopping up DNA during "import" of outside DNA. Engineers chop up DNA for further analysis.

3.2 Electrophoresis

Electrophoresis is a technique to separate and measure the lengths of a mix of DNA, RNA, or proteins. An electric field is used to drag the molecules along a gel (the shorter the molecule, the less resistance in the gel, the further the molecule advances in the gel). (see slides #9, #10)

3.3 Gel-Transfer Hybridization

Gel-Transfer Hybridization is used to detect complementary sequences, eg. in search for exon/intron. First the DNA is split into 2 single strands (denaturing). Then a corresponding mRNA is added and hybridizes with a DNA single strand to a double strand. The bend out intron sequence is removed, the double strand is denatured, and the exons can be detected by electrophoresis. (see slide #13)

Techniques similar to this technique (southern blotting) are northern blotting (for RNA) and western blotting (for proteins).


The Polymerase Chain Reaction (PCR) is used to get big amounts of identical DNA used for further analysis. It is a technically cheap method of in vitro DNA replication. It starts with denaturing the "original" DNA piece (not shown on slide #15). A build-to-order primer (of ~20 bases in length known to be part of the sequence) is added. DNA polymerase starts to build the second strand starting at the primer. When this is done, the double strand is denatured again, and the cycle restarts (see slides #16,#17). The reaction uses up nucleotides and primers, while the enzymes involved (in particular, DNA polymerase) survive the process.


Sequencing is the method of determining the sequence of DNA. The Sänger method (see slide #18) is based on provoked chain termination. A single strand of to be sequenced DNA gets completed (oligonucleotide primer,...) from a pool of nucleotides. In the first reaction, a small amount of one nucleotide (eg. A) in the pool is altered in a way that it will terminate the replication when it is used. In a massive parallel process, these marked nucleotides will be eventually used on any corresponding position of the sequence. The length of all this partial replicas ending in A can be measured at single base pair precision (Electrophoresis). Three analogous reactions are performed for the other nucleotides and yield lengths as well. The DNA sequence is given by the names of the last nucleotide of the replicas in order of increasing length. The example at the bottom of slide #18 shows the sequence ATGTCAGTCCAG.

The process of building partial replicas and reading the lengths can be automated. The altered nucleotides are distinctly marked, and the 4 types of partial replicas are build in one process. The replicas "race" in one gel-lane and the sequence is determined by laser-reading the (noisy) marks (coded as colors) of the nucleotides along the lane. (see slides #19, #20)


Originally, cloning referred to creating a replica of something, while it now is also used in a different sense. One example was described (slide #21).

Small DNA rings (plasmids) are opened using Restriction. A new piece of DNA is annealed to the two sticky ends. The result is a new, altered plasmid as well as unwanted n- (original), 2n-, 3n, ...-sized rings. The construct can be amplified or translated into proteins in the bacterium E. Coli; there are also techniques for testing whether the insertion was successful. Slides #22, #23: The plasmid contains a residence gene (AmpR), so that the presence of the plasmid in E. Coli can be determined by checking for this resistance. If LacZ' is inoperative, the insertion was successful.

Topics in Bioinformatics

diagram of topics
$Id: class3-notes.html,v 1.8 2001/01/16 06:59:49 ksjuse Exp $
Kai S. Juse
Last modified: Tue Jan 16 12:31:30 PST 2001