Abstract
One of the key features in the affinity maturation of antibodies is somatic hypermutation of the variable regions of immunoglobulin genes. The mutations that occur in immunoglobulin genes are detected by direct sequencing of cloned polymerase chain reaction (PCR) products. The frequencies of mutations in vivo are generally high enough to provide sufficient numbers of point mutations in order to generate large databases that can be analyzed in various ways. Recently, the mechanisms of variable (V)-region hypermutation have been studied in tissue culture systems and transgenic mice where mutation occurs at frequencies that are ∼10-fold lower than the estimated in vivo rate. Identifying mutations by brute force sequencing of PCR products in comparative studies is limiting when trying to determine if there are statistically significant differences. Here we describe a high throughput technique that can facilitate the identification of immunoglobulin V-regions that contain one or more mutations before sequencing. This technique, known as denaturing high-performance liquid chromatography (DHPLC), utilizes a standard HPLC apparatus with a column that binds double-stranded DNA (dsDNA). In this study, we have successfully detected ∼90% of previously sequenced mutated V-regions by DHPLC. Our results show that we were able to detect mutations throughout a 321-base pair (bp) region of the Ricin 45 immunoglobulin (Ig) V-region. Also, with the use of this assay, we have been able to detect mutations in multiple clones of different immunoglobulin genes.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 165-173 |
| Number of pages | 9 |
| Journal | Journal of Immunological Methods |
| Volume | 266 |
| Issue number | 1-2 |
| DOIs | |
| State | Published - Aug 1 2002 |
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Keywords
- Antibodies
- B lymphocytes
- DHPLC
- Mutation detection
- Somatic hypermutation
ASJC Scopus subject areas
- Biotechnology
- Immunology
Cite this
Mutation detection of immunoglobulin V-regions by DHPLC. / Bardwell, Philip D.; Martin, Alberto; Scharff, Matthew D.
In: Journal of Immunological Methods, Vol. 266, No. 1-2, 01.08.2002, p. 165-173.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Mutation detection of immunoglobulin V-regions by DHPLC
AU - Bardwell, Philip D.
AU - Martin, Alberto
AU - Scharff, Matthew D.
PY - 2002/8/1
Y1 - 2002/8/1
N2 - One of the key features in the affinity maturation of antibodies is somatic hypermutation of the variable regions of immunoglobulin genes. The mutations that occur in immunoglobulin genes are detected by direct sequencing of cloned polymerase chain reaction (PCR) products. The frequencies of mutations in vivo are generally high enough to provide sufficient numbers of point mutations in order to generate large databases that can be analyzed in various ways. Recently, the mechanisms of variable (V)-region hypermutation have been studied in tissue culture systems and transgenic mice where mutation occurs at frequencies that are ∼10-fold lower than the estimated in vivo rate. Identifying mutations by brute force sequencing of PCR products in comparative studies is limiting when trying to determine if there are statistically significant differences. Here we describe a high throughput technique that can facilitate the identification of immunoglobulin V-regions that contain one or more mutations before sequencing. This technique, known as denaturing high-performance liquid chromatography (DHPLC), utilizes a standard HPLC apparatus with a column that binds double-stranded DNA (dsDNA). In this study, we have successfully detected ∼90% of previously sequenced mutated V-regions by DHPLC. Our results show that we were able to detect mutations throughout a 321-base pair (bp) region of the Ricin 45 immunoglobulin (Ig) V-region. Also, with the use of this assay, we have been able to detect mutations in multiple clones of different immunoglobulin genes.
AB - One of the key features in the affinity maturation of antibodies is somatic hypermutation of the variable regions of immunoglobulin genes. The mutations that occur in immunoglobulin genes are detected by direct sequencing of cloned polymerase chain reaction (PCR) products. The frequencies of mutations in vivo are generally high enough to provide sufficient numbers of point mutations in order to generate large databases that can be analyzed in various ways. Recently, the mechanisms of variable (V)-region hypermutation have been studied in tissue culture systems and transgenic mice where mutation occurs at frequencies that are ∼10-fold lower than the estimated in vivo rate. Identifying mutations by brute force sequencing of PCR products in comparative studies is limiting when trying to determine if there are statistically significant differences. Here we describe a high throughput technique that can facilitate the identification of immunoglobulin V-regions that contain one or more mutations before sequencing. This technique, known as denaturing high-performance liquid chromatography (DHPLC), utilizes a standard HPLC apparatus with a column that binds double-stranded DNA (dsDNA). In this study, we have successfully detected ∼90% of previously sequenced mutated V-regions by DHPLC. Our results show that we were able to detect mutations throughout a 321-base pair (bp) region of the Ricin 45 immunoglobulin (Ig) V-region. Also, with the use of this assay, we have been able to detect mutations in multiple clones of different immunoglobulin genes.
KW - Antibodies
KW - B lymphocytes
KW - DHPLC
KW - Mutation detection
KW - Somatic hypermutation
UR - http://www.scopus.com/inward/record.url?scp=0036680022&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0036680022&partnerID=8YFLogxK
U2 - 10.1016/S0022-1759(02)00147-3
DO - 10.1016/S0022-1759(02)00147-3
M3 - Article
C2 - 12133633
AN - SCOPUS:0036680022
VL - 266
SP - 165
EP - 173
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -