OUR TEAM

Name: Matt Mortensen Open PCR Machine Engineer

Name: Garrett Salcido Open PCR machine engineer

Name: Ramesh Tadayon Experimental protocol planner

Name: Alanie Harmon Experimental protocol planner

Name: Alex Torres R&D Scientist

Name: Davey Rand R&D Scientist

LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.

System Design

Key Features
Clearly our group was more focused on correcting the lid-opening system for the top of the lid to the box. We focused on designing a new hook-and-latch system for the lid because we felt that the jerky nature of the current system was very inconvenient to the user. Whereas the current model is more difficult to open and yanking it can create potential for harm to be done to the DNA samples, our new design corrects for this by allowing means for a smooth and easy opening motion, and the samples are still protected because the lid is secured by a hook when closed. Considering that our other modification of making the sides thicker was a means to make assembling the device easier, it is reasonable to conclude that overall, we were focused on a greater convenience for the user by making a more user-friendly device.

Instructions
Altered instructions are only needed for the new hook-and-latch system: Instead of instructions to screw the Strike into the top, there will only be instructions to insert the hook into the latch. There will already be another hook for it to catch on the bottom of the lid. There should also be a note for contact information in the event that the hook was damaged or lost in the shipping process.

Protocols

Materials

PCR Protocol

Step 1: Denaturation by Heat
Initially, heat separates a DNA strand into two separate strands. This is allowed because the hydrogen bonds that hold the DNA together are weak and easily separable when heated.
Step 2: Annealing Primer to Target Sequence
We want to target a sequence specifically and in order to accomplish that you must use primers. Primers mark the end of the target sequence. Two primers are included in the PCR; one for each of the strands that were just separated during denaturation. The beginning of the DNA target sequence is also marked by the primers that bind (anneal) to the complementary sequence.
Step 3: Extension
After the primers bind to the complementary DNA, the temperature is raised and the enzyme Taq DNA polymerase is used to replicate the strands. The Taq DNA polymerase, active at high temperatures, facilitates the binding and joining of the complementary nucleotides. It synthesizes an identical double-stranded DNA strand. Extension begins at the 3’ end of the primer because Taq DNA polymerase synthesizes exclusively in the 5’ to 3’ direction, so the free nucleotides are only added to the 3’ end of the primer.

DNA Measurement Protocol

Turn on the excitation light on the fluorimeter. Put a smart phone in the cradle and set it up to take pictures of the slide. Place two drops of water in the middle of the first two rows of the slide using a pipette. Align the drop by moving the slide so the drop is in the middle of the black fiber optic fitting. Cover the fluorimeter with the light box while maintaining the ability to use the smart phone to take pictures.

Research and Development

Background on Disease Markers

Crohn's disease is a chronic, autoimmune inflammatory bowel disease (IBD) that causes inflammation of the digestive tract, also known as the gastrointestinal (GI) tract. Chronic, unmanaged inflammation can lead to a variety of symptoms. Researchers have also discovered genetic variations in certain regions of chromosome 5 and chromosome 10 that appear to contribute to Crohn disease risk. One area of chromosome 5, known as the IBD5 locus, contains several genetic changes that may increase the risk of developing this condition. Other regions of chromosome 5 and chromosome 10 identified in studies of Crohn disease risk are known as "gene deserts" because they include no known genes. The SNP's that are associated with this disease are rs11805303, rs10210302, rs9858542 in the BSN gene, rs17234657, rs1000113, rs10761659 and many more.- www.snpedia.com

Primer Design

Rs11805303:
Sequences:
TGCTTGCAAACAGAGAACTGTTTCCT[C]AAACGATCCACTTGCCTTTTATTAG
TGCTTGCAAACAGAGAACTGTTTCCT[T]AAACGATCCACTTGCCTTTTATTAG

Rs10210302:
Sequences:
AATACTGACTACCAGTGAACCATCTT[C]AACTACAGTGCTAGAAGCCTGACTG
AATACTGACTACCAGTGAACCATCTT[T]AACTACAGTGCTAGAAGCCTGACTG

Illustration