Biomedical subjects

D J Garling

Publications and source records attributed to D J Garling.

4 recordsLinked to original sources

PubMed1991

Rapid cycle DNA amplification: time and temperature optimization.

Rapid temperature cycling with hot air allows rigorous optimization of the times and temperatures required for each stage of the polymerase chain reaction. A thermal cycler based on recirculating hot air was used for rapid temperature control of 10-microliters samples in thin glass capillary tubes with the sample temperature monitored by a miniature thermocouple probe. The temperatures and times of denaturation, annealing and elongation were individually optimized for the amplification of a 536-base pair beta-globin fragment from human genomic DNA. Optimal denaturation at 92 degrees-94 degrees C occurred in less than one second; yield decreased with denaturation times greater than 30 seconds. Annealing for one second or less at 54 degrees-56 degrees C gave the best product specificity and yield. Non-specific amplification was minimized with a rapid denaturation to annealing temperature transition (9 seconds) as compared to a longer transition (25 seconds). An elongation temperature of 75 degrees-79 degrees C gave the greatest yield and increased yields were obtained with longer elongation times. Product specificity was improved with rapid air cycling when compared to slower conventional heat block cycling. Rapid thermal control of the temperature-dependent reactions in DNA amplification can improve product specificity significantly while decreasing the required amplification time by an order of magnitude.

Globins↗

PubMed1990

Minimizing the time required for DNA amplification by efficient heat transfer to small samples.

Hot-air temperature cycling of 1- to 10-microliters samples in glass capillary tubes can amplify DNA by the polymerase chain reaction in 15 min or less. A rapid temperature cycler of low thermal mass was constructed to change sample temperatures among denaturation, annealing, and elongation segments in a few seconds. After 30 cycles of 30 s each, a 536-bp beta-globin fragment of human genomic DNA was easily visualized with ethidium bromide on agarose gels. With rapid cycling, amplification yield depended on polymerase concentration. The time required for DNA amplification can be markedly reduced from prevailing protocols if appropriate equipment and sample containers are used for rapid heat transfer to the sample.

DNA↗

PubMed1984

Injury, nerve, and wound epidermis related electrophoretic and fluorographic protein patterns in forelimbs of adult newts.

Polyacrylamide slab gel electrophoresis and [35S]methionine fluorography were used to examine proteins in regenerating newt limbs, amputated denervated limbs, unamputated denervated limbs, and separated blastema mesodermal core and wound epidermis. A total of 27 protein electrophoretic bands were obtained from amputated limbs and 24 bands from unamputated limbs. Amputation resulted in the appearance of 4 new bands and the loss of 1 band as compared to unamputated limbs. These 5 banding differences were apparent on stained gels 3 days postamputation and were maintained through 10 weeks postamputation (complete regenerate stage). Only one band in unamputated limbs was always detectable on fluorographs, whereas virtually all of the stainable bands of amputated limbs were visible on fluorographs. Amputation clearly stimulated a marked, generalized increase in the synthesis of limb proteins. The 5 amputation induced changes were equally evident in stained gels of both innervated and denervated limbs. Amputated denervated limbs possessed a full set of fluorographic bands (including the 5 differences) through 18 days postamputation. However, denervation without amputation was not sufficient to alter the stainable banding pattern. Wound epidermis and mesodermal core both displayed the 5 banding differences and had identical banding patterns with the exception of one epidermal specific band. This band was also present in whole limb skin but was absent in unamputated mesodermal limb tissue. This was the only band of unamputated limbs that was consistently detectable by fluorography. It is concluded that amputation induces nerve independent changes in protein synthesis that are common to both mesodermal core and wound epidermis. These changes may represent preparation for cellular proliferation.

Aging↗

PubMed1979

Regenerative responses in larval axolotl limbs with skin grafts over the amputation surface.

Complete flaps of whole skin over the amputation surface of larval axolotl limbs did not prevent dedifferentiation, DNA synthesis, and mitosis. However, even by two weeks, insufficient cell division had occurred for blastema formation. In about 50% of the cases, small areas of wound epidermis formed over the amputated tip of the radius or ulna. At two weeks after amputation, mitotic figures and dedifferentiated cells which incorporated H3-thymidine were predominantly located in the vicinity of the small area of wound epidermis. Although delayed in relation to controls, these limbs regenerated. The results are consistent with the view that the wound epidermis is necessary to maintain dedifferentiated cells in the cell cycle.

Ambystoma↗