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.

Continue Deoxyribose

DNA replication continued

The other four carbons have been superimposed on the puzzle piece to show you their location.

DNA replication continued In DNA replication, nitrogenous bases (N-bases) are complementary.

H bond

Adenine (A) always binds to thymine (T) and forms two hydrogen bonds.

Carbon #1

C C

Carbon #4

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C

Carbon #2 Carbon #3

Cytosine (C) always binds to guanine (G) and forms three hydrogen bonds. The double-ringed N-bases are called purines (A,G) and single-ringed N-bases are called pyrimidines (C,T)

Carbon #5 Continue

Continue

DNA replication continued

The DNA molecule is a polymer of nucleotides.

Recall that a nucleotide is made of three components, • a phosphate functional group,

DNA replication continued

DNA replication stages: 1. Unwind the double helix 2. Maintain the strands separate 3. Synthesis of new strands 4. Formation of two dsDNA molecules

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a pentose (deoxyribose for DNA),

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and a N-base (A, T, G, or C).

This single-strand of DNA has 5 nucleotides shown.

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DNA replication continued

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DNA replication continued

Unwind the double helix DNA is a helical, double-stranded molecule. Helicases are enzymes that will unwind the double helix, preparing it for the next stage.

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Maintain the strands separated Since the two strands are complementary, they have a tendency to reunite. Singlestrand binding proteins stabilize the two strands.

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The 2 strands are kept separated

DNA strand is unwinding

DNA replication continued

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DNA replication continued 4. 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.

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 3’.

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Arrows show chemical direction of synthesis, 5’ to 3’

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Transcription continued

The other four carbons have been superimposed on the puzzle piece to show you their location.

Carbon #1

C C

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5’ 3’ Two dsDNA molecules

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.

3’

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Transcription is the synthesis of RNA on a DNA template. Three major RNA’s are produced: rRNA, tRNA, and mRNA.

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II. Transcription

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Carbon #4 Pentose

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Carbon #2 Carbon #3

Carbon #5 Continue

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Transcription continued

The RNA molecule is a polymer of nucleotides.

RNA N-bases are Adenine (A), Uracil (U), Guanine (G), and Cytosine (C).

Recall that a nucleotide is made of three components, • a phosphate functional group, •

a pentose (ribose for RNA),

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and a N-base (A, U, G, or C).

Transcription continued

This single-strand of RNA has 6 nucleotides shown.

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Continue

Transcription continued

Transcription continued 3’

5’

Transcription stages:

3’

dsDNA

1. Unwind the double helix

‘Bubble’

2. Synthesis of a new RNA strand

ssRNA 3. Separation of RNA from DNA, DNA double helix reforms

5’ 1. Unwind the double helix 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.

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3. Separation of RNA from DNA, DNA double helix reforms

Transcription continued

dsDNA

III. Translation

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.

DNA template To translate the message from nucleic acids to amino acids use the table in you lab manual (The Genetic Code).

RNA complementary strand In transcription if the DNA template reads ………… 5’ ATGC 3’ The RNA complementary strand will read………….. 3’ UACG 5’

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Translation, occurs in 3 steps: A. Initiation B. Elongation C. termination

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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’ UAC 5’.Complementary Nbases exist between the codon of the mRNA and the anticodon of the tRNA.

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Translation continued

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.

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Translation continued

C. 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.

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IV. Isolation of DNA from banana cells at home lab 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.

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Isolation of DNA continued

Isolation of DNA from banana cells.

Isolation of DNA continued

3. Stir the mixture slowly (avoid creating foam)

1. A fork is used to mash up a section (about ¼) of a banana.

Mashed 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

Mixture 4. Boil the mixture

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

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Continue Boiling mixture

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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)

7. Gently layer ethanol over the filtrate

6. Pipette 10 ml of the filtrate into a test tube

8. DNA precipitates in the interphase between the ethanol and filtrate

Ethanol layer

Examine the appearance of the extracted DNA.

DNA

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Filtrate layer

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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 …GGCC… and cuts between the G and the C producing blunt (or flush) ends. …G G C C… …C C G G…

CC… GG…

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Sticky ends Escherichia coli RY13 (bacteria) produces a restriction enzyme, EcoR I that recognizes the palindrome … GAATTC… and cuts between the G and the A producing sticky (or staggered) ends. …G A A T T C… …C T T A A G…

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…GG …CC

…G …GTTAA

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AATTC… G…

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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).

DNA electrophoresis continued

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 Continue

Agarose gel

Agarose gel Continue

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DNA electrophoresis continued Wells 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.

End of Lab Review ☺

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