Dna and Unknown Gene Essay

Submitted By ino0746
Words: 2587
Pages: 11
Open Document
Mapping and amplifying an unknown gene from the plant Arabidopsis thaliana.

Vyacheslav Yanyuk
Gene and Genome Analysis
Professor Hazen
March 9, 2013

Mapping and amplifying an unknown gene from the plant Arabidopsis thaliana.

Introduction

The purpose of this experiment was to form a gene model that defined major structural features of an unknown gene from Arabidopsis thaliana and to design PCR primers in order to amplify the gene from the genome. The derived gene map and protein sequence would then be used to predict its function in a future study. To correctly predict the promoter and protein-coding regions of the unknown gene an ab initio method found in Genscan software and a homology based approach were used. PCR primers were designed in order to amplify a region that included restriction enzyme sites. The PCR primers and DNA concentrations were tested to determine the best conditions for PCR. The amplified DNA was then purified, digested with restriction enzymes and assessed via gel electrophoresis, which confirmed the identity of the products and showed that the product was in fact the expected sequence.

Materials & Methods

Building a Working Map of our Unknown Gene:

In order to make a working map of the unknown gene, a web-based software called Web Map was used. Web Map analyzed the inputted sequence and provided a working map that included all 6 bp restriction enzyme sites and complete translations in all six frames. Two models were used to identify the protein encoded by the unknown gene; an ab initio method using Genscan software and a homology based model via GeneSeqer software. The ab initio method uses known rules about features that differ between protein-coding and other non-coding regions, thus Genscan provided the sequence of the predicted protein and the predicted coding sequence of the unknown gene. Using the homology-based model, sequences related to the gene were identified by expressed sequence tags (ESTs). Available ESTs were collected by similarity searching via BLAST to find ESTs with sequences that matched the sequence of the unknown gene. Five near perfect matches with at least a 90% similarity were collected. The EST information was then combined to form a larger contiguous cDNA sequence via CAP3 software. The contig was formed because multiple of the collected ESTs had the potential to overlap. The CAP3 program used the 5 ESTs to generate the smallest possible number of contiguous sequence. Next the EST contig sequence was used with GeneSeqer to accurately predict intron/exon boundaries. A full-length cDNA sequence for the unknown gene was found by BLAST and was compared to the contig that CAP3 formed. Then our gene was found in The Arabidopsis Information Resource (TAIR).

Designing PCR primers: Using a web-based software called Primer3, which is the standard primer picking software, we searched for an amplification region about 1000 base pairs long in an exon that had 2 restriction enzyme sites between 200 and 600 base pairs apart. BamH1 and Spe1 restriction enzymes were chosen because they came the closest to fit the requirements. The right primer was named oLKH_R, while the left primer was named oLKH_L. The left primer was 23 bp long with a Tm of 67.04°C. The right primer was also 23 bp long with a Tm of 66.94°C.

Testing PCR Conditions: The PCR primers were tested in order to determine the best conditions for PCR. The thermocycler was first programmed. The annealing temperature was calculated by subtracted 5°C from the primer with lowest Tm and rounding down to the nearest whole number. So, 5°C was subtracted from 66.94°C which equaled to 61°C after being rounded down. The PCR protocol was programmed as 30 sec. at 95°C and 32 cycles of 15 sec. at 95°C followed by 30 sec. at 61°C and 1 minute for extension. Gel electrophoresis was run for three dilutions of the genomic DNA with PCR products to determine which concentration works best. The dilutions were 1/100, 1/50,
Show More

Related Documents: Dna and Unknown Gene Essay

16s Rrna Synthesis Lab Report

(source). Despite there being billions of bacteria, they all share one common trait which is the 16S rRNA gene. The 16S rRNA gene is a universal gene in all bacteria and that is why it was chosen for this experiment. Using the 16S rRNA makes it possible to identify the species based on its gene sequence. (Clarridge, J. E.2004) The purpose of this experiment was to identify a sample of an unknown bacteria through the process of PCR amplification, gel electrophoresis, and cycle sequencing reaction. Along…

Words 532 - Pages 3

Adult-Onset Huntington Disease (HD)

adult-onset Huntington disease, which appears around a person’s thirties or forties, but a less common juvenile form may begin in a person’s early childhood. On a genetic level, it is characterized by an autosomal dominant mutation of the Huntingtin (HTT) gene, leading to the production of an abnormal Huntingtin protein that results in gradual cell damage; however,…

Words 294 - Pages 2

Essay about Genetically Modified Food

Genetically Modified Organisms Genetically Modified Organisms are designer foods that are commonly used for crop and human consumption using a made up genetic alter of DNA and genes. GMO’s have been commonly used within the food industry and have become a health issue in our world. When the DNA and genes are changed they are inserted to make the product grow quicker or bigger not using the natural cycle of growth. About 60% of foods prepacked in a normal grocery store contain genetically modified…

Words 521 - Pages 3

Designer Baby Research Paper

picked to have certain genes over others. In today’s consumer society, this science has an attractive appearance.This sounds questionable, or out of reach, but technology is highly capable of creating them and has process arranged. Designer babies are created through multiple genetic processes in which may be difficult to understand. A good start is with the process. Before Gene Splicing can occur to create the desired traits, Gel Electrophoresis is done to show the current DNA, it’s traits, and any…

Words 1587 - Pages 7

A Description of Breast Cancer Essay

specific cells. DNA is the chemical in each of our cells that makes up our genes—the instructions for how our cells function. We usually look like our parents because they are the source of our DNA. But DNA affects more than how we look. Some genes contain instructions for controlling when our cells grow, divide, and die. Genes that speed up cell division are called oncogenes. Others that slow down cell division, or cause cells to die at the right time, are called tumor suppressor genes. Certain changes…

Words 564 - Pages 3

Essay on Is the Epigenetic Roadmap Project worth our time

Alexandra Close Year 1 Life Sciences 9460750 Epigenetics. What is it and how can we use it for good? Epigenetics is the study of modifications and additions distributed across genome, which effect gene expression without changing the core genomic sequence of DNA (Handel et al, 2009). The NIH has set up the ‘Epigenetic Roadmap’ project which is being used to sequence Epigenetic maps which will show the variation of epigenetic tags across the genome. There has been a lot of money…

Words 1951 - Pages 8

Essay Chapter 12 Study Guide

Medicine 3. Identification of Mutations 4. Diagnosis of Affected Carriers 5. Gene Therapy 6. Agriculture 7. Forensic Science 8. Evolutionary Relationship 9. Bioplastics Advantages/DIsadvantages advantages : 1. Disease could be prevented by detecting people/plants/animals that are genetically prone to certain hereditary diseases, and preparing for the inevitable. Also, infectious diseases can be treated by implanting genes that code for antiviral proteins specific to each antigen. 2. Anothe…

Words 759 - Pages 4

Zaid S molecule life review paper

Molecule of Life Review A synthetic molecule called peptide nucleic acid (PNA) combines the information-storage properties of DNA with the chemical stability of a protein like backbone. Drugs based on PNA would achieve therapeutic effects by binding to specific base sequences of DNA or RNA, repressing or promoting the corresponding gene. A Triple stranded DNA is a structure of DNA in which three oligonucleotides wind around each other and form a triple helix. Ribonucleic acid (RNA) is a polymeric molecule…

Words 425 - Pages 2

Genomics 2012 Essay

the duplicate gene are preserved, but can the job of the original gene can sometimes be split between the two genes. Whereas in neofunctionalization the duplicate gene is transformed into a new gene. Genetic mutations that may have led to functional differences could have been nonsynonymous substitutions or a frame shift that could have happened because of insertion or deletion. These both could lead to some change in the amino acid sequence that would result in a change in the genes function. Subfunctionalization…

Words 812 - Pages 4

Cystic Fibrosis Research Paper

causes an uneven balance of minerals in the blood and therefore can cause many problems including fatigue, dehydration, and decreased blood pressure. Mutations within a gene called Cystic Fibrosis Trans membrane Regulator (also known as CFTR) is what causes cystic fibrosis. There are over 1800 different mutations of this single gene known to man and therefore is a very common issue within our society. For normal cells the CFTR protein conducts as a channel that lets cells discharge chloride and other…

Words 2816 - Pages 12