1. Multiple Sequence Alignment

2. Multiple Alignment- First pair Align the two most closely-related sequences first. This alignment is then ‘fixed’ and will never change. If a gap is to be introduced subsequently, then it will be introduced in the same place in both sequences, but their relative alignment remains unchanged.

3. ClustalW- Decision time Consult the guide tree to see what alignment is performed next. –Align a third sequence to the first two Or –Align two entirely different sequences to each other. Option 1 Option 2

4. ClustalW- Alternative 1 If the situation arises where a third sequence is aligned to the first two, then when a gap has to be introduced to improve the alignment, each of these two entities are treated as two single sequences. +

5. ClustalW- Alternative 2 If, on the other hand, two separate sequences have to be aligned together, then the first pairwise alignment is placed to one side and the pairwise alignment of the other two is carried out. +

6. ClustalW-Good points/Bad points Advantages: –Speed Disadvantages: –No way of quantifying whether or not the alignment is good. –No way of knowing if the alignment is ‘correct’.

7. ClustalW - Local Minimum Potential problems: –Local minimum problem. If an error is introduced early in the alignment process, it is impossible to correct this later in the procedure. –Arbitrary alignment.

8. Alignment of protein - coding DNA sequences It is not very sensible to align the DNA sequences of protein-coding genes. ATGCTGTTAGGGATGCTCGTAGGGATGCT-GTTAGGGATGCTCGT-AGGG The result might be highly-implausible and might not reflect what is known about biological processes. It is much more sensible to translate the sequences to their corresponding amino acid sequences, align these protein sequences and then put the gaps in the DNA sequences according to where they are found in the amino acid alignment.

9. Manual Alignment- software GDE- The Genetic Data Environment (UNIX) CINEMA- Java applet available from: –http://www.biochem.ucl.ac.uk Seqapp/Seqpup- Mac/PC/UNIX available from: –http://iubio.bio.indiana.edu SeAl for Macintosh, available from: –http://evolve.zoo.ox.ac.uk/Se-Al/Se-Al.html BioEdit for PC, available from: –http://www.mbio.ncsu.edu/RNaseP/info/programs/BIOEDIT /bioedit.html