Polymerase Chain Reaction

gel electrophoresis, sequencing cloning (into a plasmid) etc. Identifying pathogens Limitations: There is a possibility that a mutation can occur which will also be replicated during PCR. The DNA sequence for the target region should be known prior to PCR so it is not useful for regions in which the sequence is unknown. Ethical issues Some people are of the opinion that their DNA can be acquired by others easily. Also many believe that genetic engineering is unethical as manipulating DNA is unnatural.

DNA Fingerprinting Using Agarose Gel S. Aaron Sowards Bio 122 Lab 04 Brianna Adanitsch Jakob Lester Minhenga Ngijoi 2/21/18 Dr. Chad R. Sethman Abstract DNA fingerprinting is the process of analyzing an individual’s DNA base-pair patterns. The DNA fingerprinting lab involved identifying the suspect using Agarose Gel and Polymerase Chain Reaction. It was found that suspect two s DNA matched the crime scene DNA. This is known because suspect twos DNA traveled the same distance as the crime scene DNA. DNA Fingerprinting Using Agarose Gel Introduction In 1984 Dr. Alex Jeffreys came up with deoxyribonucleic acid (DNA) fingerprinting, which is also known as DNA profiling or DNA typing.

The STR length contrast is what is used to differentiate individuals. Gel electrophoresis then uses the STRs to create a DNA profile. The gel electrophoresis separates the STRs depending on their length and the pattern is then shown in fluorescent gel creating the profile. These profiles are then used by scientist to compare patterns between evidence and or suspects to determine a match or not a match. The probability of two people having the same amount of repeated sequences in STRs is one in billions of

Agarose gel electrophoresis is an easy and common technique of separating and analyzing DNA. The main objective of this lab is to find the sire of the offspring using gel electrophoresis. Gel electrophoresis is used in laboratories to isolate charged molecules like DNA, RNA, and particular proteins according to their specific size. The charged molecules travel through the gel when an electric current is spread across it. The electric current is applied across the gel so that the ends of the gel have a positive charge and the other end has a negative charge.

Single cell DNA Fingerprinting- Dr Ian Findlay and his colleagues first reported the flourishing development of a DNA fingerprint from a single cell in 1997. Single-cell DNA profiling is mainly helpful in rape cases, as DNA in sperm cells is extremely conserved due to it being so compressed in the protein head. There is also likely for the method in use in documents. 9. Mitochondrial DNA-Mitochondrial organelle is concerned with the making of cell energy.

It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved organisms. The purpose of genetic engineering is to eradicate the world of debilitating diseases. Although this is a noble goal, this branch of science also has many risks. Some people believe that we should care about genetic engineer because they believe genetically modified (GM) foods pose a serious health risk to society. However, the views concerning genet-ically modified food are mixed.

From invading people’s privacy, to having profiles made and used against someone’s will. All of these problems can be changed in time with the correct technology, but right now these problems exist in today’s method of DNA profiling. These are also reasons why people don’t want to have DNA profiling used for any

In two ways DNA binding protein can interact with the target DNA. It can bind to the target DNA in a sequence specific manner, where the DNA binding domain can recognize and bind to a specific sequence of the DNA, which is called recognition sequence. This type of interaction is call 'sequence specific DNA-protein interaction'. Sometimes the DNA binding domain can randomly bind to a double stranded as well as a single stranded DNA. It is called 'sequence non-specific DNA-protein interaction'.

Genomic instability causes individuals to maintain shorter cell cycles and also causes the bypassing of intracellular and immunological control systems. These factors give cancerous cells an advantage when it comes to growth and multiplying. There have been numerous research projects specifically dedicated to finding why this instability causes this in hopes of finding the underlying issues with cancer and stop it from being one of the leading causes of death. Genomic instability includes variations in small structures. These variations can include increased frequencies in mutations of base pairs, microsatellite instability, and changes in chromosome number or structure which are referred to as chromosome instability.

Controls are necessary to avoid false positive or false negative results. Joana et al. (2010) reported the extraction and detection of DNA along with a complete industrial soybean oil processing chain to monitor the presence of Roundup Ready (RR) soybean. The amplification of soybean lectin gene by end-point polymerase chain reaction (PCR) was achieved in all the steps of extraction and refining processes. The real-time PCR assays using specific probes confirmed all the results and proved that it is possible to detect and quantify GMOs in the fully refined soybean oil.

The first well was pre-loaded with DNA ladder and labeled as 1 microliter kb DNA ladder. Next one microliter of DNA was mixed with one microliter of loading dye using a pipettor and loaded into the well. The same mixing process was completed for the PCR product, using one microliter of PRC product and one microliter of loading dye. For the purposes of this experiment, the DNA product was loaded into well six and the PCR product was loaded into well seven. Initially DNA was loaded into well five, however gel was pierced so samples were moved one well to the right.

The Solid sequencing platform, produced by Technologies/Applied Biosystems (ABI), performs sequencing by ligation method. Similar like the Roche 454 library preparation, genomic double strand DNA were sheared into small pieces and ligated with two types of adatptors P1 and P2 on two ends. One end with P1 adaptor binds onto the surface of the magnetic bead and emulsion PCR takes place to amplify single nucleotide fragment. Then the oil was washed out and four fluorescent labeled di-bases probes were added into the beads mixture. By matching the 1st and 2nd position of the template by di-base probes, fluorescence was detected and the extra tail with fluorescent probe is cleaved out.

Next step is translation, in which the RNA becomes a protein, which then can act as structural units or enzymes. 2. How does DNA replicate itself? In order to replicate itself, DNA undergoes DNA replication, a process in which the DNA unwinds and splits in two. From that point on, new nucleotides are added to each of the original strands (A to T, C to G) until the result is two identical sequence copies of DNA.

The key enzyme in the process is DNA polymerase that forms the PCR product by linking individual nucleotide bases (adenine, guanine, cytosine and thymine). Short DNA fragments with sequence complimentary to target DNA are called primers; these specify the DNA sequence that has to be amplified. The PCR reaction mixture is made in a 96 well plate or test tube and placed in a thermal cycler. The thermal cycler is a machine used to amplify DNA by altering the temperature in repeated cycles using three precise pre programmed steps: Denaturation – By increasing the temperature in the thermal cycler the DNA is denatured creating separate single stranded DNA

Instructions: Answer the questions as directed. Upload the assignment prior to the beginning of your next lab section. Make sure you give yourself time to troubleshoot any issues you may have with uploading the assignment. You are responsible for uploading the correct file. Given the map of the plasmid in Figure 10-3, you should be able to predict the length of DNA fragments that will result when these digests are completed.