Biomedical Engineering Reference
In-Depth Information
Ligation-mediated PCR (LM-PCR)
LM-PCR is a method used for analyzing the sequence and charac-
teristics of short stretches of genomic DNA, such as when identifying
protein-DNA footprints in vivo. In LM-PCR, single stranded breaks in
the DNA are created, using Maxam-Gilbert reactions for example. After
conversion of a break to a blunt-end duplex, an oligonucleotide primer
(which has a linker associated with it) is ligated to the blunt end, and
PCR amplification is carried out. The product can then be detected by
Southern blotting or by visualization using ethidium bromide staining
after another round of PCR is carried out, using an internal (nested)
primer.
Methylation-specific PCR (MSP)
This method is used to determine the methylation pattern of cytosine
residues in genomic DNA. Cytosine residues are often methylated in
regions of DNA that are rich in CpG dinucleotides. Highly methylated
genes are often not expressed, and aberrant methylation patterns are
often associated with the inappropriate expression (or repression) of
genes in malignant cells. MSP is used to determine methylation patterns
of regulatory regions, such as promoters and enhancers, which often
correlate with transcriptional status of the corresponding gene. MSP re-
lies on the sequence differences between methylated and unmethylated
DNA that occurs when the DNA is treated with sodium bisulfite (bisulfite
converts non-methylated, but not methylated, cytosines to uracil). PCR
is then used to amplify the DNA using primers specific for methylated
and unmethylated DNA in the region of interest.
F.
Screening for differentially expressed
genes (DEGs)
Differential screening
What makes a lymphocyte a lymphocyte, and not a muscle or nerve
cell? What makes a B lymphocyte what it is, and not a T lymphocyte?
And what changes in gene expression occur as a cell becomes more dif-
ferentiated? These are questions that can be approached by identifying
differentially expressed genes. The identification of mRNA transcripts
that are expressed in one tissue but not another, or is expressed at
higher abundance, is a first step to understanding what makes cells at
different stages of development unique. It has been estimated that no
more than 10% to 20% of the genes in the genome may be expressed at
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