Topic 8B: DNA Fragments

Question 1

What is recombinant DNA technology?

Answer 1

transferring a fragment of DNA from one organism to another.

Question 2

Why can we transfer DNA fragments from one organism to another?

Answer 2

Because our genetic code is universal

Because transcription and translation mechanisms are pretty similar

Question 3

What are organisms called when they have transferred DNA in them?

Answer 3

Transgenic organisms

Question 4

What are the three was DNA fragments can be isolated?

Answer 4

• Using Reverse Transcriptase
• Using Restriction Endonuclease
• Using a Gene Machine

Question 5

Why are DNA fragments isolated?

Answer 5

Because in order to transfer a gene you have to first have the DNA fragments which contains that target gene.

Question 6

Outline the steps of using reverse transcriptase

Answer 6

• many mRNA molecules are complementary to target gene
• the complementary mRNA is mixed with free DNA nucleotides and reverse transciptase enzyme
• The enzyme is used to make cDNA

Question 7

Example of reverse transciptase being used

Answer 7

Insuline is made in the pancreatic cells. You need lots of this so but only have two copies so reverse transciptase is used to make cDNA from insulin mRNA

Question 8

What is a palindromic sequence?

Answer 8

A sequence consisting of antiparrallel base pairs base pairs read in opposite directions)

Question 9

Outline steps of using restrcition endonuclease

Answer 9

• the restriction endonuclease enzyme recognises the specific palindromic sequence and cut the DNA here.
• This is called a recognition sequence
• the shape of the cut recognition sequence is complementary to the enzyme’s active site
• between the recognition sequence could be the DNA and you use the enzyme to isolate the DNA fragment
• restriction endonucleas cuts DNA fragment vis HYDROLSIS reaction

Question 10

Using restriction endonucleas can produce…

Answer 10

sticky ends and straight ends

Question 11

Outline steps of using a gene machine

Answer 11

• the sequence that is required is designed
• first nucleotide is attached to it using a bead for support
• Add more nucleotides in order
• Add a protecting group
• Once complete the protecting group i s removed
• the oligonucleotide is joined to others to make longer DNA fragments

Question 12

What is a protecting group?

Answer 12

Its group that makes sure that the nucleotides are joined at the right points to prevent unwanted branching

Question 13

What is short sections of DNA called?

Answer 13

Oligonucleotides- roughly around 20 aa long

Question 14

What do you do after isolating DNA fragments?

Answer 14

You amplify it- make it into may copies.

Question 15

Identify two ways you can amplify DNA fragments

Answer 15

• in vivo (inside living organisms)

- in vitro (outside iving organismsl)

Question 16

Step 1 of in vivo cloning

Answer 16

• Vector DNA is cut open using the same restriction endonuclease enzyme
• the sticky ends of the vector are complementary to the sticky ends of the DNA fragments containing genes.
• vector DNA and DNA fragment are mixed together
• they combime together with DNA ligase
• this is called LIGATION
• recombinant DNA formed

Question 17

Step 2 of in vivo cloning

Answer 17

-Recombinant DNA is transferred into host cells

Question 18

What is a vector?

Answer 18

something that transfers DNA into the cell eg plasmid or baceriophage

Question 19

Differences between the plasmid vector and bacteriophage

Answer 19

-plasmid vector is persuaded into host cells where as the bacteriaphage’s DNA is injected into the cell

Question 20

What is the environment in bacterial host cell?

Answer 20

• placed in an ice cold calcium chloride
• makes cells more permeable
• plasmids added to mixture and heat shocked (around 42 degrees around 1/2 minutes)
• this encourages the cells to take in the plasmids

Question 21

How do you identify transformed host cells?

Answer 21

by adding marker genes in at the same time as vectors.

Question 22

Name types of marker genes?

Answer 22

Fluorescence- through UV light you can see them

Antibiotic Resistance- transformed cells will survive and grow

Question 23

Why is it important to identify transformed host cells?

Answer 23

Because only around 5% of host cells are taken up by DNA. Identified transformed cells can be grown and cloned more

Question 24

To produce proteins you need…

Answer 24

promoter and terminator regions

Question 25

What are promoter regions?

Answer 25

DNA sequences that tell the enzyme RNA polymerase when to start producing mRNA

Question 26

What are terminator regions?

Answer 26

DNA sequences that tell the enzyme RNA polymerase to stop

Question 27

Where can promoter and terminator regions be found?

Answer 27

in vectors or can be added along with fragments

Question 28

What is in vitro cloning

Answer 28

Amplifying DNA fragments outside living organisms by using polymerase chain reaction