enzyme lab.pdf

enzyme lab.pdf - LAB 8 DNA Restriction Enzymes Compiled by...

Info icon This preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
1 LAB 8: DNA & Restriction Enzymes Compiled by Nancy Castro, PhD, and Gorjana Bezmalinovic Laboratory Objectives After completing these exercises, you should be able to: 1. Understand the structure and function of the double-stranded DNA molecule. 2. Explain the significance of using restriction enzymes when isolating DNA fragments. 3. Understand what recombinant DNA is and how transgenic organisms are made. 4. Know the applications of gel electrophoresis. 5. Make an agarose gel and perform gel electrophoresis. 6. Explain what DNA fingerprinting is and its applications. Introduction DNA ( d eoxyribo n ucleic a cid) is a double-stranded molecule that twists together to create a helix. All living organisms contain DNA, which codes for genetic information. Each DNA molecule contains sequence specific regions that make up a single gene. Each gene is a segment of DNA with a sequence of nucleotides that provides the instructions for making a protein. These proteins are then used to carry out various biological functions in the body such as maintaining cellular structure, regulating metabolic pathways and catalyzing reactions. Our increased understanding of how DNA gets regulated has led to several advances in the biology field, especially in the medical, commercial and farming arenas. DNA molecules can now be manipulated and genetically modified through technology known as genetic engineering . Biochemical make-up of the DNA molecule DNA is comprised of four basic nucleotide building blocks: adenine, guanine, cytosine and thymine (Figure 1). These nucleotides are made up of a 5-carbon sugar deoxyribose and a phosphate backbone, and a ringed nitrogen-containing molecule, which makes up the nitrogenous base. The four nitrogenous bases are different, but they are all attached to the phosphate and sugar backbone. Only specific nitrogenous bases can be paired together. For instance, adenine (A) and thymine (T) have complementary base pairing , held together with two hydrogen bonds. Alternatively, cytosine (C) and guanine (G) have complementary base pairing, held together with three hydrogen bonds. T and C are single-ring structures called pyrimidines, and A and G are purines, double-ring structures. As seen in Fig. 1, DNA double helix looks very similar to a twisted ladder and the nitrogenous bases in the middle make up the rungs of the ladder. Figure 1. DNA double strand helix comprised of four basic nucleotides.
Image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
2 Restriction Enzymes and Recombinant DNA Restriction enzymes are specific enzymes that cleave DNA at specific sites along the molecule called restriction sites . These so called ‘molecular scissors’ are endogenously found in bacteria and are used as a defense mechanism to digest foreign DNA. The bacterial DNA is modified, usually by methylation, soon after DNA replication to help protect itself from being cut by its own enzymes. When a bacteriophage, a type of a virus that infects bacteria, attempts to invade the bacterial genome (tries to incorporate itself into the host DNA), the bacterial restriction enzymes cleave the viral DNA. These
Image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern