Recombinant DNA - DNA that carries a portion of the DNA from other organisms/species.

1. Recombinant DNA- DNA that carries a portion of the DNA from other organisms/species. Reporter genes are genetic markers with easily identifiable phenotypes (can easily see the gene being expressed). Selectable markers are one type of reporter gene. Selectable markers select for transformed cells (host cells that have taken up the foreign gene). A genomic library is a collection of DNA fragments that make up the genome of an organism. cDNA libraries are smaller DNA libraries and only include the genes transcribed in a particular tissue. They are made from cDNA, or complementary DNA. Polymerase Chain Reaction (PCR)-A technique that makes large number of DNA copies quicker than DNA cloning and only requires one cell of DNA to begin. *Taq polymerase is used. Restriction Fragment Length Polymorphisms (RFLPs) – RFLPs are restriction fragments that differ in length between individuals because of differences in DNA sequence. Short Tandem Repeats (STRs)-short sequences of nucleotide that are about 2 to 5 base pairs long that repeat multiple times. Bioremediation - human use of other organisms to remove contaminants from the environment. Ex: transform bacteria living near oil spills to help clean up oil spills (insert an oil-degrading gene). OVERVIEW OF BIOTECHNOLOGY

2. BIOTECHNOLOGY Vicky Chong, Jennifer Lauv, Michelle Leung Period 1&2

3. BIOTECHNOLOGY RESTRICTION ENYMES DNA LIBRARIES/ STEM CELLS PCR/GEL ELECTROPHORISIS / DNA FINGERPRINTING MIXED 100 100 100 100 200 200 200 200 300 300 300 300 400 400 400 400 500 500 500 500

4. What is a plasmid? What gene does it usually contain? Plasmids are a small supplemental circle of DNA. It is self- replicating and usually has the gene for antibiotic resistance.

5. How can the bacteria prevent the restriction enzyme from digesting its own DNA? The bacterium protects its own DNA by methylation or by not using the base sequence recognized by the enzymes in their own DNA.

6. What are two examples of genes restriction enzymes could contain? Describe how they would work. (Hint: GFP gene, Lac Z gene, amp^8 gene) -GFP gene: it’s obtained from jellyfish which then contains the bright green fluorescence -Lac Z gene: this gene has the code that codes for the enzyme that breaks down lactose. It also breaks down X-gal, which is a colorless but it forms a blue product. -Amp^8 gene: it gives the bacterium resistance against the antibiotic ampicillin

7. How do restriction enzymes work? Restriction enzymes cuts DNA at specific recognition sequences of nucleotides called restriction sites. Restriction enzymes usually cut at palindrome sequences since they have two identical active sites and cleave the two strands simultaneously. Many restriction sites create "sticky ends" because there is a short sequence of single-stranded DNA at each cut end.

8. Describe the process of creating recombinant DNA in plasmids. Restriction enzymes cut the DNA. The sticky ends can hydrogen bond to complementary sticky ends from other DNAs and the resulting recombinant DNA can be sealed with DNA ligase.

9. What is a genomic library? A genomic library is a collection of DNA fragments that make up the genome of an organism. How to Create A Genomic Library: Restriction enzymes break chromosomes into smaller pieces. Each fragment is inserted into a vector. Proliferation: The transformed cell proliferates in a selective medium (such as for antibiotic resistance). A colony of recombinant cells for each fragment of DNA is produced. Screening: A single petri dish can hold thousands of bacterial colonies and can be screened for the presence of a particular DNA sequence. DNA Hybridization: Colonies with a specific sequence can be identified with a probe that has complementary fluorescent (or radioactive) nucleotides. The petri dish and colonies are duplicated. One of the plates will be treated; the complementary fluorescent nucleotides will bind and identify the sequence of interest.

10. How do we make cDNA? (Two steps) • Process for Making cDNA: • Isolate mRNA from cells • Use reverse transcriptase (from retroviruses) to make a DNA molecule from the mRNA.

11. How is cDNA different from typical eukaryote DNA? cDNA does not contain introns (because it was made directly from mature mRNA).

12. What is the major difference between embryonic stem cells (ES) and adult stem cells? Adult stem cells are not able to give rise to all cell types in the organism, though they can generate multiple types. Embryonic stem cells are capable of giving rise to differentiated embryonic cells of any type.

13. What are induced pluripotent stem cells? Induced pluripotent stem cells are adult (differentiated) cells that have been reprogrammed to act like embryonic stem cells.

14. What does DNA fingerprinting compare? DNA fingerprinting compares band patterns between individuals. Each person has a unique pattern in the introns of their DNA because the number of repeats in the code differs for each person.

15. How does gel electrophoresis separate the DNA fragments? The electric field used in gel electrophoresis cause the negatively charged DNA to move toward positively charged side of the gel and the size of the fragments determine how fast they move across. Shorter fragments move faster and therefore farther than longer fragments.

16. What are RFLPs and what is their purpose? RFLPs are restriction fragments with different lengths between individuals. They are used to compare between individuals of the same species. RFLPs differ in length because of polymorphisms (slight differences in DNA sequences).

17. What is an alternative method to make copies of DNA without plasmids? Polymerase Chain Reaction (PCR). It only requires one cell of DNA to start and make multiple copies of a specific DNA segment.

18. State the steps of Polymerase Chain Reaction (PCR) The DNA is heated so that it will become denatured and unwind into two single stranded DNA. The DNA is cooled and DNA primers are added to define which sections of the DNA to clone. DNA polymerase that can withstand the heating and attach to the primers that is at each end of each single strand of DNA. It then makes a complementary DNA strand. The steps are repeated to double the number of DNA molecules during each repetition.

19. What does the mRNA analyzed in microarray technology represent? Active genes

20. In this procedure involving microarrays,what is the missing step? Take a tissue sample. Extract the mRNA from the cells. Labeled the mRNA. Spread the labeled mRNA on the microarray. ? Scan the microarray with a computer. DNA Hybridization or mRNA binds to DNA. The labeled mRNA molecules move around the microarray trying to find a match. The labeled mRNA sticks to the perfect complementary DNA on the microarray at a certain spot.

21. What are selectable markers? Selectable markers are genes used to select for transformed cells (host cells that have taken up a foreign gene).

22. What are two examples of vectors? Plasmids and viruses are vectors. The harmful genes of a virus can be deleted. The virus will still attach to a host and inject its DNA. Since viruses infect cells naturally, they have an advantage over plasmids, which don't always enter cells.

23. What is bioremediation? Bioremediation is human use of other organisms to remove contaminants from the environment. For example, bacteria have been used in wastewater treatment to break down human wastes, paper products, and household chemicals. It has also been used to transform bacteria living near oil spills to help clean up oil spills (insert an oil-degrading gene).