What is this DNA stuff?
If you've never heard of DNA, first read http://en.wikipedia.org/wiki/Dna and maybe pick up a textbook for the basics.
The critical things you should understand before trying to do this tutorial:
- DNA is a chemical entity, but we represent its sequence in the computer
- DNA is made of 4 deoxyribonucleotides, A, T, C, and G (casually called bases) in a specific sequence determined by covalent bonds
- DNA molecules have directionality; one end is the 5' terminus, the other end is the 3' terminus
- By convention, DNA sequences are always written out in the 5' to 3' direction unless stated explicitly with "5'-" and "3'-" Alternatively, DNAs can be represented in cartoon form as a line with a barb at one end. The barb refers to the 3' end.
- DNA can be circular or linear
- DNA can be single stranded or double standed
- The two molecules of a double standed DNA bond to each other by Watson-Crick base pairing
- The sequence of the complementary strand of a double standed DNA is the "reverse-complement" of the other strand
- The "reverse" and "complement" operations on a DNA sequence do not result in biochemically-meaningful sequences. You must always do both (reverse-complement) to get the sequence of the complementary strand
- With some exceptions, bacteria can only replicate double stranded circular DNAs. Genomic and plasmid DNA is therefore circular and double stranded.
- Oligonucleotides are linear single-stranded DNAs
- PCR products are linear double-stranded DNAs
- Even when DNAs are circular double stranded molecules, we represent them as linear single-stranded sequences using our software tools like ApE
Understanding the "reverse-complement" operation on a sequence is an essential concept that you will be using throughout this tutorial. If it isn't entirely clear yet, practice writing out a DNA sequences on a piece of paper, fill in what would be the complementary strand, and put in 5' and 3' ends to everything. Spin the piece of paper around and get used to the basic logic operations at play here. When you feel comfortable, proceed!