A Comparison of Hydraulic and Laser Capture Microdissection Methods for Collection of Single B Cells, PCR, and Sequencing of Antibody VDJ

During the development of B lymphocytes, a series of gene rearrangements assemble the sequences that encode immunoglobulin heavy and light chains (VDJ). Earlier studies of VDJ sequence diversification during expansion of cells in splenic or appendix germinal centers used hydraulic micromanipulation...

Full description

Saved in:
Bibliographic Details
Published in:Analytical biochemistry 2002-07, Vol.306 (1), p.55-62
Main Authors: Obiakor, Harold, Sehgal, Devinder, Dasso, Joseph F., Bonner, Robert F., Malekafzali, Arash, Mage, Rose G.
Format: Article
Language:English
Subjects:
Animals
Antibody Diversity - genetics
Appendix - metabolism
B cells
B-Lymphocytes - cytology
B-Lymphocytes - metabolism
Base Sequence
Cell Separation - instrumentation
Cell Separation - methods
Dissection - instrumentation
Dissection - methods
DNA Primers
Humans
Immunoglobulin Heavy Chains - genetics
Immunoglobulin Light Chains - genetics
immunoglobulin VDJ
Lasers
Mice
microdissection
Molecular Sequence Data
Polymerase Chain Reaction
Rabbits
Sequence Analysis, DNA - methods
single-cell PCR
Spleen - metabolism
Water
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:During the development of B lymphocytes, a series of gene rearrangements assemble the sequences that encode immunoglobulin heavy and light chains (VDJ). Earlier studies of VDJ sequence diversification during expansion of cells in splenic or appendix germinal centers used hydraulic micromanipulation (HM) to collect single B cells for PCR amplification of rearranged antibody heavy and light chain genes. PCR products were directly sequenced without a cloning step. Hydraulic micromanipulation is a very tedious method. Once capability to collect single cells by laser capture microdissection (LCM) was developed, we modified previous tissue staining and fixation methods so that we could collect cells from a given stained tissue section by HM and LCM and directly compare our success rates using these two methods. Cells were alkaline lysed and after two rounds of nested PCR products were recovered and directly sequenced. Because each rearrangement of genomic DNA that occurs to form the immunoglobulin heavy-chain-encoding sequence in developing B cells is unique, this system allowed us to verify our success rate in recovering single lymphocytes from tissue sections and amplifying a single allele. The methods developed have now made LCM an efficient alternative to HM for the collection of single B cells.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2002.5671