Rapid detection of immunoglobulin gene somatic hypermutation using heteroduplex formation

Nicholson, I.; Zola, Heddy

doi:10.1016/0161-5890(94)90071-x

Cited by 1 publication

(2 citation statements)

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“…The product gave a band at 241 bp and was analysed for mutations using the heteroduplex method. [25] Cloned V H 6 amplified product (5 ml) was mixed with unmutated V H 6 DNA (5 ml) which had been amplified with identical primers (VH6/ ND and FWR3-anti). Loading buffer (5 ml) was added to the mixture and the conditions thereafter were as described above.…”

Section: Heteroduplex Analysis Of Amplified V H 6 Dnamentioning

confidence: 99%

“…The second round primers ( Fig. 1) amplified the V H 6 region using an internal V H 6-specific primer (VH6/ND; 5Ј-CCTGTGCCATCTCCGGG-GACAGTG-3Ј; [24]) and a consensus primer complementary to the final 20 bases of the germ-line V H 6 sequence (FWR3-anti; 5Ј-ACAGTAATACACAGCCGTGT-3Ј; [25]). First round DNA products were diluted 1 : 10 in sterile water before addition to the second round reaction.…”

Section: Polymerase Chain Reaction Amplification Of Rearranged V H 6-mentioning

confidence: 99%

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Somatic hypermutation of immunoglobulin genes in human neonates

Ridings

Nicholson

Goldsworthy³

et al. 1997

Clinical and Experimental Immunology

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SUMMARYThe antibody response in the young infant is limited in several ways; in particular, responses generally are of low affinity and restricted to IgM. This raises the question whether the affinity maturation process, consisting of somatic mutation of immunoglobulin genes coupled with selection of high-affinity variants, is operative in the neonate. Re-arranged V H 6 genes were amplified by polymerase chain reaction (PCR) from cord blood and from peripheral blood of infants. Heteroduplex analysis detected mutation in only 2/18 cord blood samples, while mutations were seen from about 10 days of age onwards. Cloning and sequencing of mutated neonatal V H 6 genes showed that mutated sequences contained relatively few mutations (one to three mutations per sequence) compared with published values of about 10 in adult IgM sequences. Selection was not evident in the majority of neonatal samples. Thus mutation can occur in the human neonate, but is minimal and generally not accompanied by selection. The age at which affinity maturation develops effectively is yet to be defined.

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Section: Heteroduplex Analysis Of Amplified V H 6 Dnamentioning

confidence: 99%

Section: Polymerase Chain Reaction Amplification Of Rearranged V H 6-mentioning

confidence: 99%