chapter 7 part 3

Question 1

polymerase chaine reaction (PCR)

Answer 1

automated version of DNA replication that produces millions of copies of a short target DNA segment

Question 2

is PCR carried out in small or large volumes

Answer 2

small (less than 100 ul)

Question 3

PCR requirements

Answer 3

• double-stranded DNA template w/ target sequence
• 4 DNA nucleotides
• heat-stable DNA pol
• 2 dif single-stranded DNA primers
• buffer solution

Question 4

Taq polymerase

Answer 4

Thermus aquaticus DNA polymerase used for PCR
- lives in hot springs

Question 5

PCR steps

Answer 5

1. denaturation - reaction heat to 95 C to denature DNA into single strands
2. primer annealing - temp reduced to 45-68 C to allow primers to hybridize to complementary sequences
3. primer extension - temp raised to 72 C to allow Taq pol to synthesize DNA

Question 6

realtime-PCR/qPCR

Answer 6

modification to traditional PCR
- use spectrophotometer in each well to measure DNA product production in real-time
- use detection methods

Question 7

most common detection method for realtime-PCR

Answer 7

fluorescent dyes like SYBR Green

Question 8

benefits of realtime PCR

Answer 8

• can be used to quantitate starting amount of template DNA present accurately
• reduces detection time and uses less concentrated DNA/RNA templates
• doesn’t require DNA electrophoresis to detect products after amplification

Question 9

ddPCR

Answer 9

newest version of PCR
- creates 10s of 1000s of nanoliter-sized droplets, each containing all the components of a PCR reaction
- PCR performed in each droplet
- amount of dNA in each nano droplet read
- analyze results

Question 10

benefits of ddPCR

Answer 10

• single sample generates 10s of 1000s of data points, increasing stat power and allowing for high levels of quantitation within a single sample
• highly quantitative and very sensitive
• unparalleled precision

Question 11

Sanger sequencing development

Answer 11

Frederick Sanger in 1977

Question 12

what is Sanger sequencing also known as

Answer 12

dideoxynucleotide DNA sequencing (dideoxy sequencing)

Question 13

Sanger sequencing

Answer 13

uses DNA polymerase to replicate new DNA from single-stranded template
- 4 standard dNTP bases present in large amounts
- each reaction contains small amount of 1 dideoxynucleotide (ddNTP)
- separate reaction carried out for A, T, G, and C using corresponding small amount of one particular ddNTP
- reaction tubes produce series of partial DNA molecules
- all 4 reactions run side by side on DNA gel to determine complete sequence
- newer technologies use fluorescently laced ddNTPs and run all 4 in same lane

Question 14

ddNTP

Answer 14

lacks 3’- OH group
- can’t be added onto (chain terminates)
- replication “poison”

Question 15

what happens when a ddNTP is incorporated into the product DNA molecule

Answer 15

replication ceases

Question 16

how is a DNA gel read

Answer 16

from bottom to top

Question 17

automated DNA sequencing

Answer 17

• single reaction for each DNA sequence (all 4 ddNTPs included)
• each ddNTP labeled w/ unique fluorescent marker
• DNA synthesized, mixture of fragments produced and run on gel
• labels have dif wavelengths, laser light excites fluorescent tags
• wavelength read and recorded by computer

Question 18

next generation DNA sequencing

Answer 18

• hundreds of thousands to millions of DNA fragments in parallel
• decrease in cost, increase in speed
• identifies sequence of DNA strand during synthesis rather than by chain termination method of dideoxy sequencing

Question 19

third-generation sequencing and NGS differ from Sanger by:

Answer 19

1. using PCR to amplify DNA to allow feasible sequencing of repetitive DNA
   - massively parallel, millions of sequencing reads in each run