MODX - POLYMERASE CHAIN REACTION

PCR

Invented by Kary Mullis. An in vitro replication procedure that results in amplification of the target DNA.

0.25 mM each primer

Directs DNA synthesis to the desired region

0.2 Mm each dATP, dCTP, dGTP, dTTP

Building blocks that extend the primers

50 mM KCl

Monovalent cation (salt), for optimal hybridization of primers

10mM Tris pH 8.4

Buffer to maintain optimal pH for the enzyme reaction

1.5 mM MgCl2

Divalent cation, required by the enzyme.

2.5 units polymerase

Extends the primers (adds dNTPs)

10-10 copies of template

Sample DNA that is being tested

Specimen collection

Nucleic acid isolation

Sample assessment

PCR amplification

Visualization of result

Traditional PCR workflow

Thermus aquaticus

taq polymerase

Pyrococcus furiosus

pfu polymerase

Mg2+

Cofactor for Taq polymerase, stabilizing agent for annealing

PCR buffer

Resist pH changes

DNA template

Phage, viral DNA; plasmid, genomic DNA; cDNA. High quality and purified. Coming from a reference DNA source. 1 ng plasmid DNA. 100 ng genomic DNA

DNA primers

Short nucleotide sequence that is paired with one strand of DNA and provides a free 3’-OH end at which the DNA polymerase starts synthesis of a DNA chain. Must be specific for the gene to be amplified.

18-25 nucleotides

Primers are ideally ___ in length

40-60%

Primer Ideal GC content

2 deg difference from each other

Tm of primer pair must have a maximum of ____ difference

50-70C

Tm of primer pair must be within ______

>0.005% w/v

SDS inhibitory concentration

>0.2% (v/v)

Phenol inhibitory concentration

>1% (v/v)

Ethanol and isopropanol inhibitory concentration

>5 mM

Sodium acetate inhibitory concentration

>25 mM

Sodium chloride inhibitory concentration

>0.5 mM

EDTA inhibitory concentration

Denaturation

Separates H bond

Annealing

Allow primer to form complementary base pairing

Extension

Elongation of primers

Electrophoresis

Technique used for the separation of DNA, RNA, or protein molecules according to their size and electric charge using electric current applied to a gel matrix.

Net charge of molecule

Size and shape of molecule

Strength of electric field

Properties of the supporting medium

Temperature of operation

Rate of migration

Agarose gel

Gel for nucleic acid (cell wall of red algae Genus Gelidiaceae-tengusa and Gracilaria-ogonori)

Polyacrylamide gel

Gel for smaller nucleic acids

SDS-polyacrylamide

Gel for denaturing proteins

Running buffer

Common electrophoresis buffer

DNA stains

Serves as intercalating agents to which it may retard rate of migration of the DNA. Ethidium bromide, GelRed, and SYBR Green

18-25 Bp for general applications

Primer length for general appilcations

Primer length

Determines the specificity and significantly affect its annealing to the template.

Too short length

Low specificity, resulting in non specific amplification

Too long length

Decrease the tempalte binding efficiency at normal annealing temperature due to the higher probability of forming secondary structures such as hairpin

Melting temperature

The most important factor in determining the optimal PCR annealing temperature.

50-70c

Preferred melting temperature

Tm = 4 (G+C) + 2 (A+T)

Wallace rule

37-70C

Annealing temperature gradient

Primerselect

Analyses a template DNA sequence and chooses primer pairs for PCR and primers for DNA sequencing.

DANSIS max

Fully integrated program that includes a wide range of standard sequence analysis features

Primer Primer 5

Primer design for windows and power macintosh

Primer Primer

Comprehensive primer design for windows and power macintosh

NetPrimer

Comprehensive analysis of individual primers and primer pairs

Array designer 2

For fast effective design of specific oligos or PCR primer pairs for microarrays.

AlleleID 7

Design molecular beacons and TaqMan probes for robust amplification and fluorescence in real time PCR.

GenomePRIDE 1.0

Primer design for DNA arrays/chips

Fast PCR

Ready to use template for many PCR and sequencing applications; standard and long PCR inverse PCR. Degenerate PCR directly on amino acid sequence. Multiplex PCR

OLIGO 7

Primer analysis software for Mac and Windows

Primer designer 4

Will find optimal primers in target regions of DNA or protein molecules, amplify leatures in molecules, or create products of a specificied length.

GPRIME

Software for primer design

Sarani gold

Genome Oligo Designer. Software for automatic large scale design of optimal oligonucleotide probes for microarray experiments.

PCR Help

Primer and template design and analysis

Genorama chip design software

Complete set of programs required for genotyping chip design. The programs can also be bought separately.

Primer designer

Features a powerful, yet extremely simple, real time interface to allow the rapid identification of theoretical ideal primers for your PCR reactions

PrimerDesign

DOS-program to choose primer for PCR or oligonucleotide probes

Sequence alignment

Way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences.

Global alignment

Based on Needleman-Wunsch algorithm. Input: Treat the two sequences as potentially equivalent. Goal: identify conserved regions and differences.

Local alignment

Based on smith-waterman algorithm. Input: the two sequences may or may not be related. Goal: see whether a substring in one sequence aligns well with a substring in the other.

Dot matrix method (old method)

Dynamic programming algorithm (advanced method)

Word or k-tuple methods

Three primary method of producing pairwise alignments

Dynamic programming method

This method was first used for global alignment of sequences by Needleman-Wunch algorithm and for local alignment by Smith-Waterman algorithm. It is useful in aligning nucleotide sequence of DNA and amino acid sequence of proteins coded by that DNA.

Dynamic programming

Technique of solving optimization problems. Standard is first used on all pairs of query sequence and then the alignment space is filled in by considering possible matches or gaps at intermediate positions, eventually constructing an alignment essentially between each two sequence alignment

Global alignment

Two sequences to be aligned are assumed to be generally similar over their entire length. Alignment is carried out from beginning to end of both sequences to find the best possible alignment across the entire length between the two sequences. Applications: 1. Comparing two genes with same function. 2. Comparing two proteins with similar function

Local alignment

Does not assume that the two sequences in question have similarity over the entire length. It only finds local regions with the highest level of similarity between the two sequences and aligns these regions without regard for the alignment of the rest of the sequence regions.

Applications

1. Searching for local similarities in large sequences

2. Looking or conserved domains or motifs in two proteins.

Local sequence alignment

Subsequence comparison between a DNA sequence and a genome. Protein function domains. Exons matching

PAM

Designed to track evolutionary origin of proteins

BLOSUM

Designed to find conserved regions of proteins.