Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking

Genome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3′-ends. Upstream of the palindromes there is a degenerate sequence (8–12 nucleotides long); defined adapters are present at the 5′-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods.

Reliability of anisotropic conductive joints for bumpless flip-chip on flex packages under thermal stresses

The reliability of ACF (Anisotropic conductive film) interconnection is a serious concern especially under thermal loading condition. This paper focuses on the online contact resistance behavior of the ACF joint for bumpless flip-chip on flex packages during different thermal cycling conditions. In this work, flip chips of 11×3 mm2 having bare aluminum pad were used. Real time contact resistance (i.e. live measurement contact resistance variation with temperature) was measured by four points probe method when the packages were inside thermal shock chamber. Tests for three different thermal cycling profiles (125°C to −55°C, 140°C to −40°C and 150°C to −65°C) were carried out. The samples bonded at temperature 180°C and pressure of 2.42Mpa was used. The initial contact resistance of the bumpless samples was 0.4Ω. Contact resistance increased with the number of thermal cycles, however the effect was severe when the temperature variation was above the glass transition temperature (Tg) of the ACF matrix (131°C). Differences in co-efficient of thermal expansion (CTE) between the chip and the substrate generated thermal stresses during temperature fluctuation, which caused the pad of the substrate to slide over the Al pad of the chip. Thus variation of the contact resistance was also observed along the interconnection position in the package, i.e. corner joint showed higher value of increase in contact resistance than the middle position. Even though flex substrate was used in this study; the sliding effect was severe at the corner Al pads of the chip, where cumulative forces generated due to the thermal stress. Results show that for thermal cycling profile 140°C to −40°C, online contact resistance increased to 1.2 Ω in corner joint, whereas for the middle joints the contact resistance just increased to 0.5 Ω. Glass transition temperature (Tg) of the ACF material plays an important role on the high temperature contact resistance. For every thermal cycling profile, there is an incubation period that would have significant impact in the application of ACF. After the incubation period the contact resistance increases rapidly and the joints are no longer reliable.