Biomedical Engineering Reference
In-Depth Information
immediately by a name for the sequence with no length restriction.
Many sequences can be listed in the file, the format indicating a
new sequence at each “>” symbol found. It is important to press
Enter at the end of each line after “>” to help FastPCR recognize
the end and beginning of the sequence and the sequence name. It
is important that the first line of each sequence starts with “>”.
Degenerate DNA sequences are accepted as IUPAC code [
11
],
an extended vocabulary of 11 letters, which allows the description
of ambiguous DNA code. Each letter represents a combination of
one or several nucleotides: M = (A/C), R = (A/G), W = (A/T),
S = (G/C), Y = (C/T), K = (G/T), V = (A/G/C), H = (A/C/T),
D = (A/G/T), B = (C/G/T), N = (A/G/C/T), U = T, and I
(Inosine). The program accepts amino acid codes: A (Ala), C
(Cys), D (Asp), E (Glu), F (Phe), G (Cly), H (His), I (Ile), K
(Lys), L (Leu), M (Met), N (Asn), P (Pro), Q (Gln), R (Arg), S
(Ser), T (Thr), U (Sec), V (Val), W (Trp), and Y (Tyr).
3.5 Alignment
Format Description
There are many different programs that perform different types of
alignment formats. Standardizing on a set of formats enables
programs to be written that can read results from many different
programs. In all alignment formats, gaps that have been introduced
into the sequences to make them align are indicated by the “-”
character. The exception to this rule is GCG/MSF format which
uses “.” as the gap character inside the sequences. The file may
begin with as many lines of comment or description as required.
The first mandatory line must contain the text “MSF,” “Alignment
as simple alignment format,” “DIALIGN,” or “MEGA” to be
recognized as alignments from these programs. Following the first
line are lines that start with the sequence name, which is separated
from the aligned sequence residues by white space.
4
The PCR Primers or Probe Design Analysis Options
4.1 PCR Primer
Design Generalities
Primer design is one of the key steps for successful PCR. For PCR
applications, primers are usually 18-35 bases in length and should
be designed such that they have complete sequence identity to the
desired target fragment to be amplified. The parameters, either
controllable by the user or selected automatically, are primer length
(12-500 nt), melting temperature for short primers calculated by
nearest neighbor thermodynamic parameters, theoretical primer
PCR efficiency (quality at %) value, primer CG content, 3′ end
terminal enforcement, preferable 3′ terminal nucleotide sequence
composition in degenerated formulae, and added sequence tags at
5′ termini. The other main parameters used for primer selection
are the general nucleotide structure of the primer such as linguistic
complexity (nucleotide arrangement and composition), specificity,
