Chapter 19 Biotechnology

Question 1

The effect of size of the DNA molecules on gel electrophoresis

Answer 1

Smaller molecules move faster through the gel than larger ones

Question 2

The effect of molecular charge of the DNA molecules on gel electrophoresis

Answer 2

Negatively charged molecules move faster than positively charged ones since they move toward the positive end of the gel. More applicable for proteins, because proteins have differing levels of charge.

Question 3

The effect of molecular shape of the DNA molecules on gel electrophoresis

Answer 3

Tightly condensed globular molecules migrate more quickly than linear molecules (has to do with secondary structure)

Question 4

A reaction developed in 1983 that is used for exponential synthesis of a specific target DNA

Answer 4

Polymerase Chain Reaction (PCR)

Question 5

Sequences in PCR that are complementary to sequences that flank the target sequence, from where synthesis continues

Answer 5

Primers

Question 6

The most commonly used polymerase for PCR, originally isolated from Thermus aquaticus, a eubacteria that thrives in hot springs, has a lower accuracy rate than the DNA polymerase in replication and lacks 3’-5’ exonuclease activity (proofreading)

Answer 6

Taq Polymerase

Question 7

The first step of PCR, in which the sample is heated to 90-100 degrees C to separate the target DNA into single strands

Answer 7

Denaturation

Question 8

The second step of PCR in which the temperature is reduced to 30-65 degrees C to allow the primers to hybridize to their complementary sequences

Answer 8

Primer Annealing

Question 9

The third step of PCR, in which the temperature is increased to 72 degrees C to allow DNA synthesis

Answer 9

Primer Extension

Question 10

The phase of PCR amplification in which the reaction components are in excess and there is exact doubling of the amount of DNA at each cycle. This is also the phase of Real-time PCR quantification

Answer 10

Exponential Phase

Question 11

The phase of PCR amplification in which the reaction components are being consumed and amplification slows down

Answer 11

Linear Phase

Question 12

The phase of PCR amplification in which the reaction components are depleted and the amount of DNA does not increase. Also the phase where endpoint detection, which is not quantitative and only serves to determine whether the DNA is present or not, determine the size of the amplified DNA target, or to amplify DNA for use in experiments, takes place.

Answer 12

Plateau Phase

Question 13

A type of PCR in which the amount of DNA is quantified at each cycle and plotted against cycle number, used to accurately quantify the amount of template. This is done in a thermocycler with a fluorescence detector

Answer 13

Quantitative PCR (qPCR)

Question 14

A type of qPCR which uses two oligonucleotides (the forward and reverse primers) as well as a dye that binds to double stranded DNA but not single stranded DNA, resulting in to fluorescence at each denaturing step.

Answer 14

SYBR Green

Question 15

A type of qPCR that uses three oligonucleotides (the forward primer, reverse primer, and a probe with a fluorophore at the 5- end and a quencher at the 3’ end). When Taq Polymerase reaches the probe, it uses it’s 5’-3’ exonuclease activity to break the probe, seperating the fluorescent chemical from the quencher, therefore activating the fluorescence

Answer 15

TaqMan Assay

Question 16

A sequencing method in which four seperate sequencing reactions occur per sample, each with one ddNTP in a 4:1 ratio with the corresponding dNTP. The ddNTPs will be incorporated at random, and when they are, the synthesis will stop, producing DNA of different lengths that can be put on a gel to get the final sequence.

Answer 16

Sanger (Dideoxy) Sequencing

Question 17

The sequencing “generation” with Sanger Sequencing

Answer 17

First generation Sequencing

Question 18

The sequencing “generation” with NGS and Illumina Sequencing

Answer 18

Second generation Sequencing

Question 19

The sequencing “generation” with single-molecule real-time (SMRT) sequencing

Answer 19

Third Generation Sequencing

Question 20

A type of sequencing that is much faster and cheaper, with a lot of samples sequenced simultaneously

Answer 20

Massively Parallel Sequencing (MPS)

Question 21

Sequencing that uses four fluorescently-labeled nucleotides to sequence millions of clusters on the surface of a flow cell.

Answer 21

Sequencing by Synthesis

Question 22

The terminators used in Illumina Sequencing that have a fluorophore and a block at the 3’ position that can be washed away to allow sequencing to continue

Answer 22

Reversible Terminators

Question 23

A unit of repeat that s 1-6 bp long and can be found repeated five to fifty. There can be many alleles of these in a population, which are produced due to strand slippage during DNA replication.

Answer 23

Short Tandem Repeats (STRs or Microsatellites)

Question 24

The identification of individuals through DNA analysis

Answer 24

DNA fingerprinting