For the purpose of
DNA replication
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
, the HpaII tiny fragment Enrichment by Ligation-mediated
PCR Assay (HELP Assay)
is one of several techniques used for determining whether DNA has been
methylated
In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These t ...
. The technique can be adapted to examine DNA methylation within and around individual
genes
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
, or it can be expanded to examine methylation in an entire
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
.
The technique relies upon the properties of two
restriction enzymes
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
: ''Hpa''II and ''Msp''I. The HELP assay
compares representations generated by ''Hpa''II and by ''Msp''I digestion of the genome followed by ligation-mediated PCR. ''Hpa''II only digests 5'-CCGG-3' sites when the cytosine in the central CG dinucleotide is unmethylated, the ''Hpa''II representation is enriched for the hypomethylated fraction of the genome. The ''Msp''I representation is a control for copy number changes and
PCR amplification difficulties.
It was recently shown that cytosine methylation patterns tend to be concordant over short (~1 kb) regions. The patterns represented by the ''Hpa''II sites therefore tend to be representative of other CG dinucleotides locally.
The analysis of HELP data involves quality analysis and normalization. An analytical pipeline written in the
R programming language
R is a programming language for statistical computing and graphics supported by the R Core Team and the R Foundation for Statistical Computing. Created by statisticians Ross Ihaka and Robert Gentleman, R is used among data miners, bioinform ...
was recently published to allow HELP data processing.
References
External links
The protocol for the HELP assay
{{DEFAULTSORT:Help Assay
Microbiology