DNA Technology

Question 1

vectors

Answer 1

Things used to transport genes into cells

Question 2

first vectors used were

Answer 2

plasmid vectors

Question 3

plasmid vectors

Answer 3

contain only a few genes (non essential)

bacteria will take in plasmids from the environment

Question 4

what takes in plasmids

Answer 4

only bacteria take in plasmids, not eukaryotes

Question 5

virus vectors

Answer 5

viruses inject DNA into nucleus of host cell
We replace virus DNA with genes we want placed in a cell
used in gene therapy

Question 6

example of what virus vectors treated

Answer 6

B in B disorder (no immune system, live in sterile environment)
virus put new gene in stem cells
stem cells returned to bone marrow
10 of 11 cured (3 later got leukemia)

Question 7

restriction enzymes

Answer 7

Enzymes that cut DNA ONLY at specific sites

only cuts double stranded DNA

Question 8

how do restriction enzymes cut at specific sites

Answer 8

Sites are identified by nucleotide patterns on both strand of DNA

Question 9

Restriction enzymes were isolated from

Answer 9

Bacteria, which use them as primitive immune system to cut virus DNA

Question 10

cDNA

Answer 10

complimentary DNA

Question 11

why cDNA

Answer 11

used to put human gene in bacteria
bacteria can’t remove introns from mRNA transcripts so we remove introns from the gene before we give it to them
we let the human cell do transcript processing
then use mRNA as a template

Question 12

making cDNA

Answer 12

1. Get human mRNA for needed protein
2. Add reverse transcriptase that bonds DNA nucleotides to RNA
   this DNA is the cDNA
   3.Add DNA polymerase to remove RNA nucleotides and replaces them with DNA nucleotides
3. End result = double stranded DNA
   can be cut with restriction enzymes
   & added to plasmids that bact. take in.

Question 13

probe

Answer 13

used to located gene of interest, Short strand of DNA complementary to gene of interest, Tagged with radiolabel or tracer, binds to gene of interest which makes hybrid DNA

Question 14

Hybrid DNA

Answer 14

Hybrid DNA = any DNA where the 2 sides of the double helix come from different sources

Question 15

DNA isolation

Answer 15

isolate the gene you want from the rest of the genome

Question 16

DNA isolation process

Answer 16

A. Use probes to label DNA 
B. Cut DNA using restriction enzymes
C. Separate DNA using electrophoresis
D. Use only DNA that includes the radiolabeled probe
E.Reduces volume of unwanted DNA

Question 17

electrophoresis

Answer 17

reduces volume of unwanted DNA
separates DNA by size
uses agar and electricity, DNA moves toward positive end, little pieces go the fastest

Question 18

primer

Answer 18

A. Short, synthetic, single strand of RNA or DNA
B. Complimentary to DNA in front of gene of interest
C. Initiates DNA synthesis

Question 19

PCR

Answer 19

polymerase chain reaction, makes thousands of copies

Question 20

Step A for PCR

Answer 20

Mix DNA containing GoI with

1) primers for target gene
2) spare nucleotides (A, T, G, C)
3) DNA Polymerase from Taq polymerase which is a heat resistant enzyme

Question 21

Step B for PCR

Answer 21

Step B : heat DNA strands to break H-bonds between base pairs and separate the strands

Question 22

Step C for PCR

Answer 22

cool DNA so base pairs re-bond ( some bond to primer)

Question 23

Step D for PCR

Answer 23

repeat whole process, 1) mix DNA with primers, nucleotides, and taq

2) heat to break H bonds
3) cool to bond to primer
4) repeat

Question 24

Gel Electrophoresis-

Answer 24

separates DNA based on size of fragment

Question 25

electrophoresis process

Answer 25

Samples of DNA cut with same restriction enzymes
Dye added to visualize DNA
Samples placed in wells of gel
Gel placed in liquid buffer (to run current through)
Electric current run through buffer & gel
DNA moves through the gel to positive end
smaller pieces move faster

Question 26

Factors affecting DNA movement in gel electrophoresis

Answer 26

1. More porous gel (less dense) speeds up movement

2. Higher current speeds up movement

Question 27

gel analysis

Answer 27

A. DNA standard marker of known length are in lane 1
B. Measure distance each segment moved
C. Graph distance vs size
D.Best fit line
E. Estimate unknown size

Question 28

DNA fingerprinting

Answer 28

Different people have different lengths of DNA between genes

Question 29

the Differences in human genes are in the

Answer 29

non-coding DNA between genes tandem repeats, the number of repeats varies, so the lengths of DNA varies

Question 30

DNA fingerprinting uses

Answer 30

Crime scene investigations

Paternity tests

Question 31

paternity tests

Answer 31

child gets all DNA from mom or dad, so Any fragment found in child must be in either Mom’s DNA or Dad’s DNA

Question 32

DNA sequencing

Answer 32

Alter nucleotides so they will stop replication

Question 33

DNA sequencing process

Answer 33

Label Nucleotides with fluorescent dye each base different color
Alter labeled nucleotides so they stop DNA replication
Add both labeled nucleotides and normal nucleotides to sample DNA
(Add modified A nucleotides to one sample,T to second, G to third, C to fourth)
Run PCR then electrophoresis
Order of segments indicates order of bases in sequence

Question 34

Sanger Method (dideoxy method)

Answer 34

method for DNA sequencing, Uses Dideoxynucleotide to stop replication, named after Frederick sanger, won nobel prize in chemistry

Question 35

reading DNA sequencing

Answer 35

Segments separated by size

Base that fluoresces is always the last base in line

Question 36

GMO

Answer 36

genetically modified organism

Question 37

genetic engineering

Answer 37

foreign genes added to organisms

Question 38

GMO example (bt gene from Bacillus thuringiensis)

Answer 38

bt gene from Bacillus thuringiensis

 a) bacterial protein toxic to insects
 b) used by WHO to kill mosquitoes
 c) used by organic farmers
 d) added to corn potato cotton so plant makes its own bt protein insecticide
 e) specific to borers & bollworm

Question 39

bt mechanism

Answer 39

plant uses bt gene to make protein, insect eats protein, protein binds to receptors in gut, gut wall breaks down, only insects with receptor affected, studies show no harm to Monarchs

Question 40

bt advantages

Answer 40

less pesticide, more specific delivery

Question 41

transgenic

Answer 41

organism with foreign DNA

1) DNA microinjection into egg
2) all cells of organism have new DNA