BME100 f2017:Group2 W0800 L4

Section 1: Protocol Planning

Goal:

A Plan For Polymerase Chain Reactions: The first step is to extract the DNA and moving it into a special PCR tube. PCR works by Heating and cooling the solution several times. After Primer 1 is added to the PCR tube. Primers attach to sites on the DNA strands that are at either end of the segment you want to copy. Then add Primer 2 which will attach to the second site. Then Nucleotides are added to the PCR tube. These are the genetic building blocks that will be used to create billions of DNA copies. The final step in the process is that DNA polymerase will be added to the PCR tube. The DNA polymerase is special for this experiment because it is able to with stand the high amount of heat from the reaction.

Lab​ ​scenario​:​

​Several​ ​patients​ ​have​ ​submitted​ ​DNA​ ​to​ ​be​ ​tested​ ​for​ ​a disease​ ​marker.​ ​Your​ ​group​ ​will​ ​receive​ ​three​ ​replicate​ ​DNA​ ​samples from​ ​two​ ​patients*.​ ​The​ ​results​ ​from​ ​all​ ​BME100​ ​teams’​ ​tests​ ​will eventually​ ​be​ ​collected​ ​on​ ​a​ ​master​ ​chart​ ​for​ ​statistical​ ​analysis.​ ​If​ ​you mix​ ​up​ ​or​ ​mislabel​ ​your​ ​samples,​ ​you​ ​could​ ​misdiagnose​ ​a​ ​patient.

Materials:

• Lab​ ​coat​ ​and​ ​disposable​ ​gloves
• PCR​ ​reaction​ ​mix,​ ​8​ ​tubes,​ ​50​ ​μL​ ​each:​ ​Mix​ ​contains​ ​Taq​ ​DNA​ ​polymerase,​ ​MgCl​2​,​ ​and​ ​dNTP’s (http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless -master-mix-m714-protocol/
• DNA/​ ​primer​ ​mix,​ ​8​ ​tubes,​ ​50​ ​μL​ ​each:​ ​Each​ ​mix​ ​contains​ ​a​ ​different​ ​template​ ​DNA.​ ​All​ ​tubes have​ ​the​ ​same​ ​forward​ ​primer​ ​and​ ​reverse​ ​primer
• A​ ​strip​ ​of​ ​empty​ ​PCR​ ​tubes
• Disposable​ ​pipette​ ​tips:​ ​only​ ​use​ ​each​ ​only​ ​once.​ ​​Never​ ​reuse​ ​disposable​ ​pipette​ ​tips​.​ ​If​ ​you​ ​do, the​ ​samples​ ​will​ ​become​ ​cross-contaminated
• Cup​ ​for​ ​discarded​ ​tips
• Micropipettor
• OpenPCR​ ​machine:​ ​shared​ ​by​ ​two​ ​groups

OpenPCR Program:

• Heated Lid: 100°C
• Initial Step: 95°C for 2 minutes
• Number of Cycles: 25 denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 Seconds
• Final Step: 72°C for 2 minutes
• Final Hold: 4°C

Section 2: Research and Development

Patient ID #s: 13955 and 93490

Q1: Components of PCR reaction Template DNA: The specific DNA sequence that you want copy/replicate. Primers: Short oligonucleotides of DNA, usually around 8-60 base pairs in length. Taq Polymerase: A thermostable DNA polymerase, Taq cannot begin DNA replication without the addition of a starting primer. Deoxyribonucleotides (dNTP’s)​: 4 amino acids Adenine (A), guanine (G), cytosine (C), and thymine (T). They are needed to provide the building blocks for DNA replication

Q2: What happens to the components (listed above) during each step of thermal cycling? INITIAL STEP: 95°C for 2 minutes: The DNA strands are heated to near boiling and the two strands straighten out. Denature​ at 95°C for 30 seconds: The separated DNA strands separate completely Anneal​ at 57°C for 30 seconds: Primers bind to the target DNA sequence and bracket the region of DNA that is to be copied. Extend​ at 72°C for 30 seconds: The Taq Polymerase binds to the end of the primer site and matches complementary nucleotides to the template DNA and  start to extends that strand. FINAL STEP: 72°C for 2 Minutes: The Taq polymerase fully extends the DNA strands from the primer to form new double-helix strands of the target DNA. FINAL HOLD: 4°C: Short term storage of the newly amplified DNA strands

DNA is made up of four types of molecules called nucleotides, designated as A, T, C and G. Base-pairing, driven by hydrogen bonding, allows base pairs to stick together. Which base anneals to each base listed below? Adenine (A) to Thymine (T) and vice versa.  Cytosine (C) to Guanine (G) and vice versa.

Q4. During which two steps of thermal cycling does base-pairing occur? Explain your answers. Base pairing occurs between the Annealing and Extension. After the two DNA strands have been denatured into single strands, the samples are cooled down to 57°C. The short DNA primers bind to the target in the newly created single-stranded DNA. The temperature is raised slightly to 72°C so that the Taq Polymerase binds to the end of the primer, matches the base pairs along the DNA. After new nucleotides are formed.

Section​ ​1:​ ​Disease​ ​SNP-specific​ ​primer​ ​design

What is a nucleotide? A nucleotide is an organic compound made up of a nitrogenous base, a sugar, and a phosphate group. It is the basic building block of nucleic acid polymers, such as DNA and RNA. The sugar group of a nucleotide may be ribose or deoxyribose. The ribose sugar is the sugar component that makes up RNA and the deoxyribose sugar is the sugar component that makes up DNA.

What is polymorphism? Genetic polymorphism is used by geneticists and molecular biologists to describe mutations in the genotype, such as single nucleotide polymorphisms. It is used to describe multiple forms of a single gene that exists in an individual or among a group of individuals. It refers to multiple forms of a gene that can exist. (example being an earlobe; it can be either attached or not attached.) This is not like height, which is a continuous variation.

What species is the variation found in? Latin name- homo sapiens.

What Chromosome is the variation located on? 19:44907853

What is listed as the clinical significance of this SNP? Pathogenic-virus, bacterium, or microorganism causing disease. In the Pubmed link with summaries of research associated with SNP. What condition is linked to SNP? Screening and evaluation of Deleterious SNPs in APOE Gene of Alzheimer’s Disease.

What does APOE stand for? APOE stands for Apolipoprotein E.

What is the function of APOE? APOE link/gene ontology/first three unique terms. Amyloid-beta binding, amyloid-beta binding (second term), antioxidant activity… cholesterol binding.

What is an allele? One of two or more alternative forms of a gene that are made from a mutation and are located at the same location on the chromosome. These DNA codings determine distinct traits that can be passed on from parents to offspring. (example being genes that exist in two allele forms, such as AB blood type inheritance, and multiple alleles for a trait, such as ABO blood type.) The disease associated allele contains what codon? The non-disease allele= CTG, a change in this allele at the T position creates the disease associated allele that contains the codon CCG. Numerical position of the SNP: 44907853

SNP Information and Primer Design

Single nucleotide polymorphisms (also known as SNPs) are a single change in a lone nucleotide which is the basic building block unit of the human DNA, (nucleotides are attached to a sugar phosphate backbone to create a double helix DNA shape). SNPs are quite common and occur all of the time, however every once in awhile the mutation can cause serious negative consequences including deadly diseases. SNPs happen nearly once in every 300 nucleotide chain, and often times can be a great biomarker. Whenever the SNP happens to occur within what is known as the “regulatory region” or within the gene this can change the gene’s function and ultimately the protein. This is how a SNP can cause a change that leads to destruction of the normal healthy function of the human body. However, scientists are currently using SNPs to give a heads up to people about what kind of diseases that person is more inclined to develop. Also, SNPs can play a role in deciding what kind of medicine is most impactful on a patient, and what certain medicines would be of no help. The allele that is associated to the disease in this example contains the codon CCG, and the non-disease allele contains the codon CTG. A different codon ultimately leads to a different amino acid brought along by the tRNA, and finally a mutation in the protein that could be very costly.

Primer Design and Testing

This certain species is a costly variation that is found in Homo sapiens, and the chromosome where the specific variation is found is given the numerical position of 44907853. The conditions that are more readily developed due to the increase in lifespan thanks to new medical advances are induced by this SNP. These conditions are known as alzheimer's disease and strokes, all thanks to the codon change. Part 3: Numerical position of the non-disease reverse primer- 44908053 Non-disease​ ​reverse​ ​primer​ ​​: 5’- C A G G C C C C C C A A G A C T T A G C 3’ Non disease forward primer- 5’A G C G G C C A G C G C T G G G A A C T 3’ Disease​ ​forward​ ​primer​ : 5’-​ ​A G C G G C C A G C G C T G G G A A C C 3’