Chapter 9

Question 1

Genetic engineering

Answer 1

Altering an organisms genetic information using in vitro techniques

Question 2

Polymerase chain reaction (PCR)

Answer 2

The presence of a specific segment of DNA can be detected, and the size determine, in only a matter of hours. Can diagnose infectious disease if DNA specific to pathogen can be amplified

PCR product: can be visualized via gel electrophoresis

Question 3

Probe technologies

Answer 3

Colony blots are used to detect companies that contain a specific DNA sequence; flourescence in situ hybridization is used to identify cells directly in a specimen

Question 4

Restriction enzymes

Answer 4

Cut DNA strands a recognition sequence

Generates restriction fragments

Allows creation of recombinant DNA molecules

Recognize 4 to 6 base pair nucleotide sequence

Question 5

Palindromes

Answer 5

The same on both strands when read in 5’ to 3’ direction

Question 6

DNA Gel electrophoresis

Answer 6

Separates DNA fragments by size

Current causes DNA to migrate through gel (+) electrodes; smaller fragments move faster

Dye reveals visible bands of DNA fragments

Question 7

CRISPR systems

Answer 7

Can locate an altar specific sites in a DNA molecule

Cas9- used gene editing
Dead Cas9- doesn’t cut DNA, RNA guide still binds to DNA

Single-stranded RNA recognizes specific DNA sequence

Question 8

Genetically engineered bacteria

Answer 8

Useful for:

Protein production

DNA production

Research tools

CRISPR/Cas9- engineering can DNA clone

Question 9

DNA cloning

Answer 9

Isolate DNA

Cut with restriction enzyme

Join insert (DNA) with vector (plasmid) to generate recombinant molecule

Introduce into host for replication

High copy number vector makes large amounts of protein

Transgenic: a plant or animal with a cloned gene

Question 10

GMOs

Answer 10

Insulin cloned into bacteria

Cheese production: chymosin (rennin)

Vaccine production : hepatitis B, foot and mouth disease

Question 11

DNA library

Answer 11

Collection of clones that together contain the entire genome

Restriction enzymes

Clone all fragments

Question 12

Generating a recombinant DNA molecule

Answer 12

Vector is usually modified plasmid or bacteriophage

Origin of replication; carriers cloned DNA

Let’s have restriction site where DNA is cut so that insert can be joined to it

Selectable marker: A gene encoding resistance to antibiotics

Second marker is disrupted by intersectional inactivation when insert is present

Question 13

Vector pUC18

Answer 13

Selects for sales with vector and differentiates those with recombinant plasmids

Selectable marker: ampicillin resistance

Second genetic marker: lacZ gene

Question 14

Recombinant DNA advisory committee (RAC)

Answer 14

Test new technologies for safety

Numerous benefits, but potential for malicious use (bioterrorism)

Question 15

DNA sequencing

Answer 15

Determining the order of nucleotides in a DNA molecule

Question 16

Human genome project

Answer 16

Lead to new techniques used to sequenced genome of numerous organisms—-> advances in genomics

Question 17

Dideoxy chain termination

Answer 17

In vitro DNA synthesis

Requires:
Template DNA
DNA polymerase
Primer

Dideoxynucleotides (ddNTPs) act as chain terminators

Laser reads fluorescent labels on separated ddNTPs

Question 18

High throughput sequencing

Answer 18

Next generation methods

Highly automated; fast, lower costs

Errors are common so genomes are analyzed multiple times

Question 19

Nanopore sequencing

Answer 19

Used to sequence microbial DNA on international space station

Long fragments of ssDNA in electrically conductive solution, and Passthrough microscopic poor wear different nucleotides black electric current to varying degrees

Disruptions reflect nucleotide sequence

Question 20

Reverse transcription PCR (RT-PCR)

Answer 20

Reverse transcriptase used to synthesize cDNA from mRNA tablet in a sample

cDNA is template for amplification

Question 21

Quantitative PCR (qPCR)

Answer 21

Tracks amplification in real time and determines relative amount of target DNA in sample, Fluorescent marker

Also called real-time PCR

Question 22

DNA synthesis requires

Answer 22

Double stranded DNA with target sequence to serve as template

Taq polymerase: heat stable DNA polymerase from Thermus aquaticus

Primers: determine length of DNA amplified, only target sequence

Deoxynucleotides

Question 23

Three-step amplification cycle

Answer 23

DNA denatured by heating

Temperature lowered to allow primers to anneal

Temperature raised to allow DNA synthesis

Question 24

Short tandem repeats (STRs)

Answer 24

STRs repeat consecutively a variable number of times

Usually within intron or other untranslated region

Question 25

DNA probes

Answer 25

Locate specific nucleotide sequence Single stranded piece of DNA complementary to the sequence of interest Anneals to complement through hybridization

Question 26

Colony blotting

Answer 26

Detect specific DNA sequence in colonies growing on agar plates

Question 27

Florescence in situ hybridization (FISH)

Answer 27

Probe hybridizes to nucleotide sequences in intact cells fixed to microscope slide Probe often binds to rRNA sequences present in high copy number Can detect related organisms or specific species with different markers

Question 28

DNA Microarrays

Answer 28

Allow study of gene expression Also called Gene chips or biochips Array has oligonucleotides that act as probes mRNA is isolated and used to synthesize labeled cDNA cDNA added to array anneals to sequences of genes that were being expressed