Unit 13: Review Biotechnology Lab

Size: px

Start display at page:

Download "Unit 13: Review Biotechnology Lab"

Terence Fowler
7 years ago
Views:

1 Unit 13: Review Biotechnology Lab I. DNA replication DNA replication is a process in which DNA makes an exact copy. DNA replicates through a semiconservative method. The chemical direction of replication is 5 (five prime) to 3 (three prime). Examine the deoxyribose (sugar) puzzle piece. Deoxyribose is a pentose, a monosaccharide containing five carbons. This puzzle piece shows only one of the carbons, carbon #5. Deoxyribose The other four carbons have been superimposed on the puzzle piece to show you their location. In DNA replication, nitrogenous bases (N-bases) are complementary. Adenine (A) always binds to thymine (T) and forms two hydrogen bonds. H bond arbon #1 ytosine () always binds to guanine (G) and forms three hydrogen bonds. arbon #4 arbon #5 arbon #2 arbon #3 The double-ringed N-bases are called purines (A,G) and single-ringed N-bases are called pyrimidines (,T) The DNA molecule is a polymer of nucleotides. Recall that a nucleotide is made of three components, a phosphate functional group, a pentose (deoxyribose for DNA), DNA replication stages: 2. Maintain the strands separate 3. Synthesis of new strands 4. Formation of two dsdna molecules and a N-base (A, T, G, or ). This single-strand of DNA has 5 nucleotides shown. 1

2 DNA is a helical, double-stranded molecule. Helicases are enzymes that will unwind the double helix, preparing it for the next stage. 2. Maintain the strands separated Since the two strands are complementary, they have a tendency to reunite. Singlestrand binding proteins stabilize the two strands. DNA strand is unwinding The 2 strands are kept separated 3. Synthesis of new strands With the exposed N-base sequence, DNA polymerase can add new DNA nucleotides to the template (in dark blue). The new strand (in light blue) is synthesized 5 to Form two dsdna molecules The result is two dsdna molecules. Now the cell can continue the process of cell division (dividing into 2 cells) because it has two copies of the DNA Arrows show chemical direction of synthesis, 5 to Two dsdna molecules II. Transcription Transcription is the synthesis of RNA on a DNA template. Three major RNA s are produced: rrna, trna, and mrna. The other four carbons have been superimposed on the puzzle piece to show you their location. Ribosomal RNA (rrna) is a component of ribosomes and involved in protein synthesis Transfer RNA (trna) is the carrier of amino acids, contains the anticodons Messenger RNA (mrna) dictates the amino acid sequence, contains the codons The chemical direction of transcription is 5 to 3. Examine the ribose (sugar) molecule of DNA. Ribose is a pentose, a monosaccharide containing five carbons. Pentose arbon #4 arbon #5 arbon #1 arbon #2 arbon #3 2

3 The RNA molecule is a polymer of nucleotides. Recall that a nucleotide is made of three components, a phosphate functional group, RNA N-bases are Adenine (A), Uracil (U), Guanine (G), and ytosine (). a pentose (ribose for RNA), and a N-base (A, U, G, or ). This single-strand of RNA has 6 nucleotides shown. Transcription stages: 3 dsdna 3 5 Bubble 2. Synthesis of a new RNA strand 3. Separation of RNA from DNA, DNA double helix reforms ssrna 5 RNA polymerase unwinds the double helix a a bubble appears. (The two blue strands separate.) 2. Synthesis of a new RNA strand (in pink) RNA polymerase brings in RNA nucleotides complementary to the DNA template. This action moves from 5 to 3 direction. 3. Separation of RNA from DNA, DNA double helix reforms III. Translation DNA template RNA complementary strand dsdna In transcription if the DNA template reads 5 ATG 3 The RNA complementary strand will read.. 3 UAG 5 After the mrna is synthesized it moves into the cytoplasm and becomes associated with the ribosome. Translation is the process of having trna molecules bring in correct amino acids which are linked to one another. To translate the message from nucleic acids to amino acids use the table in you lab manual (The Genetic ode). Translation, occurs in 3 steps: A. Initiation B. Elongation. termination 3

4 Translation continued Translation continued A. Initiation The binding of the first trna to mrna. The mrna codon is 5 AUG 3. Because it initiates translation, this codon is referred to as the start codon. According to the genetic code, the codon AUG codes for the amino acid Methionine. The trna that carries Methionine has the anticodon, 3 UA 5.omplementary N- bases exist between the codon of the mrna and the anticodon of the trna. B. Elongation The addition of as many amino acids as needed to build the polypeptide. Each amino acid is carried by the trna to the mrna. Translation continued IV. Isolation of DNA from banana cells at home lab. Termination Elongation will continue until a stop signal appears, encoded by a termination (stop) codon. There are three stop codons, UAA, UAG, UGA. When a stop codon is encountered, the peptide chain is released, the mrna and ribosomal subunits dissociated, and protein synthesis is terminated. The preparation of DNA from any cell type involves the same general steps: 1. breaking open the cell and nuclear membrane (if present), 2. removing proteins and cellular debris from the nucleic acids, and 3. purification of the DNA This procedure allows for the crude extraction of DNA from banana cells. Isolation of DNA continued Isolation of DNA continued Isolation of DNA from banana cells. 1. A fork is used to mash up a section (about ¼) of a banana. 2. In a heat-resistant vessel (i.e. Pyrex) the mashed banana is combined with 250 ml deionized water, 25 ml of a soap solution and 25 ml of meat tenderizer solution Meat tenderizer is a saturated solution. Just add tenderizer to water until it stops going into solution. Liquid hand soap solution is 50% water and 50% soap Mashed banana 3. Stir the mixture slowly (avoid creating foam) 4. Boil the mixture Mixture Boiling mixture 4

5 Isolation of DNA continued Isolation of DNA continued 5. Strain the mixture through 4 layers of cheesecloth and collect the filtrate (fluid that has passed through a filter) 6. Pipette 10 ml of the filtrate into a test tube 7. Gently layer ethanol over the filtrate 8. DNA precipitates in the interphase between the ethanol and filtrate Ethanol layer Examine the appearance of the extracted DNA. DNA Filtrate layer V. DNA electrophoresis DNA electrophoresis continued Restriction endonucleases or restriction enzymes (REs) are enzymes obtained by bacteria that physically cut DNA. REs are named according to the following guidelines. The first letter is the same letter as the first letter of the genus name of the organism from which is was isolated and is italicized. The second and third letters are the first two letter of the specific epithet and are italicized. The fourth letter, not italicized, indicates the specific strain of the organism. Roman numerals indicate the order of discovery. RE s recognize a 4- or 6-base pair sequence that is a palindrome and cut the DNA in the same way every time. REs attach to the DNA at a specific recognition sites, called restriction sites. Some cut through the complementary strands at the same position, producing blunt ends. Others cut producing sticky ends. Blunt ends Haemophilus aegyptius (bacteria) produces a restriction enzyme, Hae III that recognizes the palindrome GG and cuts between the G and the producing blunt (or flush) ends. G G GG G G + GG Sticky ends Escherichia coli RY13 (bacteria) produces a restriction enzyme, EcoR I that recognizes the palindrome GAATT and cuts between the G and the A producing sticky (or staggered) ends. G A A T T G AATT T T A A G GTTAA + G DNA electrophoresis continued DNA electrophoresis continued The resulting fragments of DNA can be separated from one another by electrophoresis. Electrophoresis is the process of applying voltage to a solution of charged molecules (DNA or proteins). An electrical field is applied causing the DNA fragments to move from their origin through the gel toward the positive electrode, since DNA is negatively charged. Loading the gel DNA fragments are placed in wells made on the agarose gel (similar to gelatin). one of the wells Agarose gel Agarose gel 5

6 DNA electrophoresis continued The DNA fragments move at different rates depending on their size. Small fragments migrate faster than larger ones. Fragments of the same size concentrate in one group forming a band (thin line) on the gel. The gel is removed from the chamber and stained. Examine the stained gel. Remember that the DNA fragments all start in the well and travel toward the right side, from the cathode(-) to the anode (+) end. Wells End of Lab Review - + 6

Molecular Genetics. RNA, Transcription, & Protein Synthesis

Molecular Genetics. RNA, Transcription, & Protein Synthesis Molecular Genetics RNA, Transcription, & Protein Synthesis Section 1 RNA AND TRANSCRIPTION Objectives Describe the primary functions of RNA Identify how RNA differs from DNA Describe the structure and