Lecture 2

Question 1

What does PCR do?

Answer 1

allows for amplification of a specific sequence of DNA

Question 2

What does electrophoresis and blotting do?

Answer 2

allows researches to find a specific macromolecule in a larger population

Question 3

What enzyme was discovered that helped to synthesize DNA in a lab? Why was it useful?

Answer 3

Taq - Thermus aquatus

had adaptations in its proteins that allowed it to be stable in a wide range of temperatures w/o denaturing

Question 4

What are the requirements for PCR?

Answer 4

• template DNA: starting material
• Primers: tell what region of DNA will be copied
• Polymerase (Taq) & Buffers: proper conditions
• dNTP’s: nucleotides
• Thermocycler: can quickly change temps (optional)

Question 5

What are the 3 steps of PCR?

Answer 5

1. Denature DNA ( 95 degrees): takes double stranded DNA - breaks apart H bonds
2. Anneal Primers ( 45-65 degrees): cold enough fr things to stick. Primer allows for faster binding of DNA
3. Synthesize new DNA ( 72 degrees)

Question 6

How many PCR cycles need to occur for the desired product?

Answer 6

three cycles

Question 7

what direction does the polymerase work in?

Answer 7

The 5’ to 3’ direction. 2 polymerase will work in opposite directions

Question 8

what is the equation to figure out the copies of the target molecule?

Answer 8

2^n-2n

Question 9

what is the equation to figure out the total copies?

Answer 9

2^n

Question 10

what does gel electrophoresis do?

Answer 10

separates DNA fragments based on size. small fragments will run through the gel faster than large ones because of the pores in the gel. The gel has a negative charge

Question 11

what are the 2 methods for sequencing?

Answer 11

1. Sanger sequencing or chain termination ( main method)

2. Chemical degradation method

Question 12

what reagents are required in Sanger sequencing?

Answer 12

• DNA to be sequenced
• polymerase (Taq)
• primers
• dNTP’s -nucleotides
• ddNTP’s - termination nucleotides

Question 13

why are ddNTP’s termination nucleotides?

Answer 13

they don’t have OH therefore they can’t add more nucleotides - stops the synthesis

Question 14

what is blotting?

Answer 14

The transfer of a macromolecule to a solid substrate after electrophoresis. it is required for further analysis of a sample

Question 15

what are the four types of blots?

Answer 15

1. Southern blot: transfer of DNA to membrane
2. Northern blot: transfer of RNA
3. Western blot: transfer of protein
4. Eastern blot: post translational modification of proteins

Question 16

what is hybridization used for in analyzing blots?

Answer 16

to identify the presence of specific molecules

Question 17

what do you do for a western blot detection?

Answer 17

• wash with antibody- binds against target proteins
• May use a secondary antibody - increases signal and binds to first antibody
• antibody may also carry some covalently linked detection molecule - where the antibody binds the signal is produced