Answer in your packets!!!  Warm-up What is Chargaff’s Rule? What are the three parts of a nucleotide? What is the significance of Purines and Pyrimidines? What is DNA’s backbone made of?

Answers What is Chargaff’s Rule? A=T and C=G What are the three parts of a nucleotide? Sugar, phosphate, nitrogen base What is the significance of Purines and Pyrimidines ? Purines = 2 parts (A & G), Pyrimidines = 1 part (T & C) They are opposite throughout the double helix (chargaff’s rule) What is DNA’s backbone made of? Sugars and phosphates

Fix the dates! Notes: stage 1, Sticky-ends DNA Activity What’s on the Agenda 3/8 (A) & 3/7 (B) Notes: stage 1, Sticky-ends DNA Activity 3/12 (A) & 3/9 (B) Notes: stage 2, Plasmid Matching 3/14 (A) & 3/13 (B) Notes: stages 3 & 4, Bacteria Activity 3/16 (A) & 3/15 (B) Notes: stage 5, GMO discussion 3/20 (A) & 3/19 (B) DNA Extraction Lab 3/22 (A) & 3/21 (B) Open-note Test on Unit 8

Agenda Warm-up: DNA Questions Lecture/Notes on DNA Technology Blunt and Sticky Ends Activity

Unit 8 DNA Technology

Remember DNA… Contains genetic info (heredity) Made of 4 nucleotides (ATGC) Purines & Pyrimidines Double Helix Two Strands Codes for Protein

Genetic Engineering AKA: Gene Modification; the manipulation of an organism’s genes by use of biotechnology. Traditional (older) methods of gene modification include: Inbreeding and selective breeding

DNA Technology Overview Isolation – of the DNA containing the targeted gene. Insertion – of the DNA into a vector. Transformation – Transfer of DNA into a suitable host. Identification – finding host organisms containing the vector and DNA (by use of gene markers). Growth/Cloning – of successful host cells.

Stage 1 – Producing DNA fragments How is complementary DNA made using reverse transcriptase? How are restriction endonucleases used to cut DNA into fragments?

Reverse Transcriptase A group of viruses called retroviruses (e.g. HIV) contain an enzyme called reverse transcriptase. It is used to turn viral RNA into DNA so that it can be transcribed by the host cell into proteins.

Reverse Transcriptase makes DNA from an RNA template – it does the opposite of transcription.

Using reverse transcriptase You choose a gene that you want. Extract mRNA from a cell that is making the gene you want. A single-stranded complementary copy of DNA (cDNA) is made using reverse transcriptase on the mRNA template. Single-stranded cDNA is used to make double stranded DNA using DNA polymerase. This forms a double stranded copy of the gene you wanted.

Normal Transcription Reverse Transcription

Restriction Enzymes Bacteria contain restriction enzymes which help to protect themselves from invading viruses. Restriction enzymes cut up viral DNA at specific sites – this property can be useful in gene technology. There are 2 ways that restriction enzymes make cuts. Blunt Ends Sticky Ends

Restriction Enzymes – “Blunt Ends” Some restriction enzymes cut straight across both DNA chains, this forms what are called blunt ends.

Restriction Enzymes – “Sticky Ends” Some restriction enzymes make a staggered cut in the two DNA chains, forming sticky ends (uneven).

Sticky Ends Sticky ends have a strand of single stranded DNA on one side which are complementary to another single strand. They will join with another sticky end only if it has been cut with the same restriction enzyme. Meaning, that its letters will match up with the other side.

Restriction Enzymes Are also know as Endonuclease. Hint: all enzymes end in “ase”. Have highly specific activation sites. Usually about 4-8 base pairs (letters). These are called recognition sites. Recognition sites are usually palindromic, which means the sequence and its complement are the same but reversed. Ex: GAATTC and the complement CTTAAG

Palindrome Examples

VOCABUARY Nucleotide Genetic Engineering Gene Modification Inbreeding Selective Breeding Retrovirus Reverse Transcription RNA mRNA DNA cDNA Transcription DNA Polymerase Gene Restriction Enzyme Blunt Ends Sticky Ends Recognition Sites

DNA Blunt & Sticky-Ends Activity In your assigned groups, work together to fit your DNA together to form two stands (with blunt ends on the outside). The colored squares can be (A, T, G, or C). Write the letter that would be in the blank box into the matching color box in your jigsaw chart. Determine the animal your DNA is from by matching the letters your wrote in the colored boxes. Example: Try a second puzzle too!

Key to how the DNA is arranged:

Warm-up What type of virus can perform transcription backwards? What type of molecule ends in “-ase”? What is the difference between blunt and sticky ends?

Answers What type of virus can perform transcription backwards? Retrovirus What type of molecule ends in “-ase”? Enzymes What is the difference between blunt and sticky ends? Blunt = flat, sticky = single strand of DNA attached

Agenda Lecture/Notes: Stage 2 Lecture/Notes: Stage 3 and 4 Plasmid Matching Lecture/Notes: Stage 3 and 4 Bacteria & Antibiotics Activity

Stage 2 – Insertion in to a vector What is the importance of “sticky ends”? How can a DNA fragment be inserted into a vector?

Importance of “sticky ends” DNA from different source can be joined together IF they have the same sticky ends = the same recognition site. In order to have the same sticky ends they must have been cut using the same restriction endonuclease. Sticky ends are joined together using a glue called DNA Ligase: it bonds the sugar-phosphate backbone together. The new DNA molecule is now called Recombinant DNA, because it was made by re-combining different DNA together.

Insertion of DNA into a vector VECTOR = something used for transport. PLASMID = the vector used to transport genes in DNA. A plasmid is a circular piece of DNA found in bacteria (not the chromosome). Plasmids are useful because they contain antibiotic resistance genes. Types of Vectors

The Bacterial Plasmid Sometime an antibiotic resistant gene is broken/cut-into when the restriction enzyme cuts open the plasmid vector to make room for inserting the targeted gene. There are many other antibiotic resistant genes that are not cut, they stay intact and are used to help select the correct host cells that the plasmid will be transferred into. If you are using a plasmid to make an antibiotic (penicillin for example) then you don’t want to put that plasmid into a cell that will be killed by penicillin, you want to put it into a cell that is resistant to penicillin (antibiotic resistant). Then the cell will produce the antibiotic and not die. The penicillin can be harvested and used to help someone in need of that medicine.

Insertion into plasmids 1- Cut the gene you want out of the DNA 2- Cut the matching sticky-end area out of a bacterial plasmid 3- Use DNA Ligase to glue the DNA gene into the bacterial plasmid 4- A hybrid plasmid is made! It contains a new gene! 5- The bacteria makes whatever the new gene coded for.

VOCABULARY Ligase Recombinant DNA Vector Plasmid Hybrid Plasmid

Vector-Plasmid Matching Place the pictures in order from the beginning to the end of the DNA-Vector-Plasmid process. One or two statements below each picture. When complete, raise your hand to have Mrs. Cooper check your work. Get the OK to glue into your packet. Answer the questions at the bottom of the activity page.