Thermal Cyclers

The instrument that heats and cools a DNA sample in order to perform the PCR reaction is known as a thermal cycler. Precise and accurate sample heating and cooling is crucial to PCR in order to guarantee consistent results. There are a wide variety of thermal cycler options available from multiple manufacturers. These instruments vary in the number of samples that can be handled at a time, the size of the sample tube and volume of reagents that can be handled, and the speed at which the temperature can be changed. Prices for thermal cycling devices range from a few thousand dollars to over 10 000 dollars.

Perhaps the most prevalent thermal cycler in forensic DNA laboratories is the GeneAmp® PCR System 9600 from Applied Biosystems (Foster City, CA). The '9600' can heat and cool 96 samples in an 8 X 12-well microplate format at a rate of approximately 1°C per second. The 9600 uses 0.2 mL tubes with tube caps. These tubes may be attached together in strips of 8 or 12, in which case they are referred to as 'strip-tubes.'

Table 43

Thermal cycling parameters used to amplify short tandem repeat DNA markers with commercially available PCR amplification kits. Cycling parameters differ because reaction components, in particular the primer concentrations and sequences, vary between the different manufacturers' kits.

aThe first 10 cycles are run with a denaturation temperature of 94°C and the last 20 cycles are run at 90°C instead. The Promega PowerPlex 1.1, 2.1, and 16 kits also use specific ramp times between the different temperature steps that differ from the conventional 1°C/second.

Table 43

Thermal cycling parameters used to amplify short tandem repeat DNA markers with commercially available PCR amplification kits. Cycling parameters differ because reaction components, in particular the primer concentrations and sequences, vary between the different manufacturers' kits.

Step in Protocol

AmpF/STR® kits (Applied Biosystems)

GenePrint® STR kits (Promega Corporation)

Initial Incubation

95°C for 11 minutes

95°C for 11 minutes

Thermal Cycling

28 cycles

30 cycles3

Denature

94°C for 1 minute

94°C for 30 seconds (cycle 1-10) 90°C for 30 seconds (cycle 11-30)

Anneal

59°C for 1 minute

60°C for 30 seconds

Extend

72°C for 1 minute

70°C for 45 seconds

Final Extension

60°C for 45 minutes

60°C for 30 minutes

Final Soak 25°C (until samples removed) 4°C (until samples removed)

Final Soak 25°C (until samples removed) 4°C (until samples removed)

Modern thermal cyclers use a heated lid to keep the PCR reagents from condensing at the top of the tube during the temperature cycling. However, there are some forensic laboratories that still use an older model thermal cycler called the DNA Thermal Cycler '480'. Samples amplified in the 480 require an overlay of a drop of mineral oil on top of the PCR reaction mix in order to prevent evaporation since there is no heated lid.

Thermal cyclers capable of amplifying 384 samples or more at one time are now available. The Dual 384-well GeneAmp® PCR System 9700 can run 768 reactions simultaneously on two 384-well sample blocks. Thermal cyclers capable of high sample volume processing are valuable in production settings but are not widely used in forensic DNA laboratories.

Was this article helpful?

0 0
Stammering Its Cause and Its Cure

Stammering Its Cause and Its Cure

This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.

Get My Free Ebook


Responses

  • doroteo
    How are thermocyclers are used in forensic science?
    9 years ago

Post a comment