Biotech

Question 1

What is Biotech?

Answer 1

• Humans modifying DNA
• Used to make protein products for medicine, agriculture, etc

Question 2

What is Recombinant DNA?

Answer 2

DNA made in a lab by combining DNA fragments from multiple sources

Question 3

How many biotech tools are there? What are they?

Answer 3

1. Restriction Enzyme
2. Plasmids
3. PCR
4. Gel Electrophoresis
5. Dideoxysequencing
6. CRISPR (clustered regulatory interspaced short palandromic repeats)

Question 4

State Restriction Enzymes Process

ALLOWS RECOMBINANT DNA TO BE FORMED

Answer 4

1. RENs (restricion endonuclease) cut double stranded DNA at specific recognition sites (4-6bps palindromes)
2. RENS leave the cuts as blunt ends or sticky ends (overhang)
3. To keep bacteria cell’s own DNA from being cut, bacterial cell adds methy groups to own restriction sites using enzyme methylase
4. Cloning occurs (restriction sites are used to insert DNA into other cells and make copies)
   5.** Ligase** seals phosphodiester bonds to close the cut.

Question 5

What is A Plasmid?

Answer 5

• small, circular double stranded DNA
• replicate in host cell
• independant but may use RNA polymerase and ribosomes in host cell to express their own proteins
• Mobile

Question 6

Plasmid

Answer 6

1. Vector: carrier of DNA segment into host cell for cloning or recombinant DNA technique
2. Cells get transformed from taking up new DNA using heat shocks or electroporation

Question 7

What do Engineered plasmids have?

Answer 7

1. Origin of replication
   * so plasmid can replicate
2. Restriction Site(s)
   * Desired gene cen be inserted. Plamids cand be cut using RENs
   * Selectable marker (Way to check if cells have taken up plasmids; resistant ones will grow).

Question 8

PCR

POLYMERAE CHAIN RXN

Answer 8

1. Mimics DNA replication

Question 9

What is in PCR

Answer 9

• DNA primers
• Free nucleotides
• target sequence DNA to copy
• buffer
• heat stable polymerase (taq polymerase)

Question 10

What is the Process of PCR?

Answer 10

1. DENATURING:Double stranded DNA gets denatured using heat (94-96). (H-bonds break and make single strands)
2. ANNEALING: DNA primers attach to 3’ ends. Temp lowers to 50-65 so DNA anneals
3. EXTENDING: Taq polymerase attaches to primers, synthesizing strands.

Question 11

What is Gel Electrophoresis

Answer 11

• Analyzes pieces of DNA based on size (lenght in bps)
• DNA might be cut using RENs
• more copies made by PCR

Question 12

Process of Gel Electrophoresis

Answer 12

1. Gel is made with agarose and put with buffer solution
2. fill gel wells using micropipette with DNA, dye and visualizing chemical
3. Have one well with the ladder (DNA fragment sizes that are known)
4. Turn on power and watch DNA move to cathode
5. Look at results usinng U.V light and compare with the ladder

Question 13

What is in Gel Electrophoresis?

Answer 13

• Buffer
• Gel with the wells (made with agarose)
• Power source
• tray with anode (-) and cathode (+)
• DNA sample with dye and visualizing chemical (ethidium bromide)

Question 14

What is Dideoxy Sequencing?

Answer 14

Used to find the exact sequence of base pairs (A, T, C, G) in a gene

Question 15

What is in Dideoxy Sequencing? (Dideoxy Sanger method)

Answer 15

• Template: A fragment to be sequenced
• 4 types of diddeoxy nucleotides (ddA, ddT, ddC, ddG)
• 4 tubes: One for A, T, C, G
• Primers, enzymes, regular deoxynucleotides, etc.

Question 16

What are dideoxy analogues?

Answer 16

Missing OH group on its 3’ carbon in nucleotide. DNA replication will stop cuz DNA polymerase adds to 3’OH

Question 17

What is The Process of Dideoxy Sequencing?

Answer 17

1. 4 tubes of the dideoxy nucleotide is used and DNA replication occurs.
2. when dd nucleotide is incorporated, replication stops forming multiple different DNA fragments.
3. For Ex: ddA tube contains:
   * ATCG
   * ddA
   * template: TAGCCATG
   * replicated: ATCGGTAC
4. Ran on electrophoresis gel so sequence can be read (shortest to longest|cathode to anode)