4.5 DNA Cloning

Question 1

What has our knowledge of DNA biology led to?

Answer 1

an ability to manipulate the genes of organisms

Question 2

What is a genome?

Answer 2

the complete genetic makeup of an organism

Question 3

What is genetic engineering?

Answer 3

practice of cloning genes and then using them to alter the genome of viruses and cells

Question 4

What are the uses of genetic engineering?

Answer 4

ranges from producing a product to treating genetic disorders

Question 5

What is cloning?

Answer 5

the production of identical copies of an organism through asexual means

Question 6

Why are human identical twins also considered clones?

Answer 6

because the first two cells of the embryo separated and each became a complete individual

Question 7

What is gene cloning?

Answer 7

the production of many identical copies of a single gene

Question 8

Why do biologists clone genes?

Answer 8

• want to produce large quantities of the gene’s protein product such as human insulin
• learn how a cloned gene codes for a particular protein
• use the genes to alter the phenotypes of other organisms in a beneficial way

Question 9

What is gene therapy?

Answer 9

when cloned genes are used to modify a human

Question 10

What are transgenic organisms?

Answer 10

organisms with foreign DNA or genes inserted in them

Question 11

What are transgenic organisms frequently used for?

Answer 11

to produce a product desired by humans

Question 12

What do most processes rely on to produce cloned DNA?

Answer 12

recombinant DNA technology and the polymerase chain reaction

Question 13

What is recombinant DNA (rDNA)?

Answer 13

contains DNA from 2 or more different sources such as the human cell and the bacterial cell

Question 14

What do researchers need to make rDNA?

Answer 14

vector

Question 15

What is a vector?

Answer 15

a piece of DNA that can be manipulated such that foreign DNA can be added to it

Question 16

What is plasmid?

Answer 16

a common vector that are small accessory rings of DNA from bacteria that are not part of the bacterial chromosome and are capable of self-replicating

Question 17

How were plasmids discovered?

Answer 17

by investigators studying the bacterium Escherichia coli

Question 18

What are the two types of enzymes that are used to introduce a foreign DNA into vector DNA?

Answer 18

restriction enzyme: cleaves the vector DNA

DNA ligase: seals DNA fragments

Question 19

Why are both DNAs cleaved with the restriction enzyme?

Answer 19

so they can have complementary ends that can anneal

Question 20

Where do hundreds of restriction enzymes occur naturally?

Answer 20

in bacteria. where they act as a primitive immune system by cutting up any viral DNA that enters the cell

Question 21

Why are they called restriction enzymes?

Answer 21

because they restrict the growth of viruses

Question 22

What else can restriction enzymes be used for?

Answer 22

as molecular scissors to cut double-stranded DNA at a specific site

Question 23

Why is both foreign DNA and vector DNA cleaved with the same restriction enzyme?

Answer 23

a gap exists into which a piece of foreign DNA can be placed if it ends in bases complementary to those exposed by the restriction enzyme

Question 24

What are “sticky ends”?

Answer 24

the single-stranded but complementary ends of the two DNA molecules

Question 25

Why are they called "sticky ends"?

Answer 25

they can bind a piece of foreign DNA by complementary base pairing

Question 26

What do "sticky ends" facilitate?

Answer 26

the insertion of foreign DNA into vector DNA

Question 27

What is DNA ligase used for?

Answer 27

to seal the foreign piece of DNA into the vector

Question 28

What do bacterial cells do?

Answer 28

take up recombinant plasmids, especially if the cells are treated to make them more permeable

Question 29

What happens when the plasmid replicates?

Answer 29

so does the foreign DNA and thus the gene is cloned

Question 30

What are the steps of gene cloning? (4)

Answer 30

1. restriction enzyme cleaves DNA 2. DNA ligase seals human gene and plasmid 3. host cell takes up recombined plasmid 4. gene cloning occurs

Question 31

Describe cloning a human gene.

Answer 31

- human DNA and bacterial plasmid DNA are cleaved by a specific type of restriction enzyme (ie. human DNA containing the insulin gene is spliced into a plasmid by DNA ligase) - gene cloning is achieved after a bacterium takes up a plasmid - if the gene functions normally as expected, the product (ie. insulin) may also be retrieved

Question 32

What is DNA fingerprinting?

Answer 32

a technique that involves treating the entire genome with restriction enzymes

Question 33

Why does each person have a unique collection of DNA fragment sizes?

Answer 33

because each person has their own restriction enzyme sites

Question 34

What is gel electrophoresis?

Answer 34

where an electrical current is used to force DNA through a porous gel material

Question 35

What is a DNA fingerprint?

Answer 35

each person's unique collection of DNA fragments are separated according to their size during gel electrophoresis smaller fragments move farther through the gel than larger fragments and results in a pattern of distinctive bands called a DNA fingerprint

Question 36

What is short tandem repeat profiling?

Answer 36

the method of choice STRs are the same short sequence of DNA bases that recur several times as in GATAGATAGATA

Question 37

Why is STR profiling advantageous?

Answer 37

does not require the use of restriction enzymes and instead, PCR is used to amplify target sequences of DNA which are fluorescently labelled