Recombinant Dna Technology

Front 1

What is recombinant DNA technology?

Back 1

Technology that allows DNA to be manipulated, altered and transferred from one organism to another

Front 2

How is this technology possible?

Back 2

The DNA code is universal, so the genetic code is the same in all organisms. Also, transcription and translation are essentially the same in all organisms

Front 3

What are the stages of making a protein using DNA technology?

Back 3

Isolation

Insertion

Transformation

Indentification

Growth/cloning

Front 4

What occurs in isolation?

Back 4

The DNA fragments with the gene for the desired protein are isolated

Front 5

Which methods can be used to isolate the DNA fragments?

Back 5

Conversion of mRNA to cDNA

Using restriction endonucleases

Using the gene machine

Front 6

How can we isolate DNA from mRNA?

Back 6

We can extract mRNA from the cell's cytoplasm as it's more abundant than DNA. We can use the mRNA as templates to make lots of complementary DNA using reverse transcriptase.

Front 7

What then happens to the cDNA?

Back 7

It's single stranded, so DNA polymerase is used to build up the other strand with complementary nucleotides, giving us double stranded DNA

Front 8

Where are restriction endonucleases found and what are they used for?

Back 8

They're found naturally in all organisms. They're used as a defensive mechanism against pathogens. The host can use them to cut u viral DNA.

Front 9

What does this mean?

Back 9

There's a large variety of restriction endonucleases that can cut DNA at a specific palindromic sequence known as a recognition sequence

Front 10

What does palindromic sequence mean?

Back 10

The sequence is the same backwards a forwards

Front 11

What is the first type of cut restriction endonucleases can make?

Back 11

Cuts between opposite base pairs, leaving 2 straight edges known as blunt ends

Front 12

What is the other type of cut they can make?

Back 12

They can cut in a staggered fashion, so each strand of DNA has exposed, unpaired bases known as sticky ends.

Front 13

What is step 1 of making DNA with a gene machine?

Back 13

The desired sequence of nucleotide bases of a gene is determined from the amino acid sequence of the desired protein. mRNA codons for each amino acid are looked up and complementary DNA triplets re worked out

Front 14

What are steps 2 and 3 of making DNA with a gene machine?

Back 14

The desired base sequence is fed into a computer. The gene is then checked for biosafety and biosecurity to ensure it meets international and ethical standards.

Front 15

What are steps 4 and 5 of making DNA with a gene machine?

Back 15

The computer designs a series of small overlapping single strands of nucleotides (oligonucleotides) which can be assembled into the gene. Oligonucleotides are assembled by adding one nucleotide at a time in the require sequence.

Front 16

What is step 6 of making DNA with a gene machine?

Back 16

The oligonucleotides are joined together to make a gene without introns. This is replicated by PCR

Front 17

What is step 7 of making DNA with a gene machine?

Back 17

PCR is used to construct the complementary strand of nucleotides to make the double stranded gene

Front 18

What is step 8 of making DNA with a gene machine?

Back 18

Using sticky ends, the gene can then be inserted into the vector.

Front 19

What is step 9 of making DNA with a gene machine?

Back 19

The genes are checked using standard sequencing techniques and errors are rejected.

Front 20

What happens in insertion?

Back 20

The DNA fragment is inserted into a vector

Front 21

What is step 1 of insertion?

Back 21

The DNA fragment is cut out of the host DNA using a restriction endonuclease producing sticky ends. This is done twice

Front 22

What is step 2 of insertion?

Back 22

Vector DNA e.g. plasmid is isolated and cut with the same enzyme once

Front 23

What is step 3 of insertion?

Back 23

The sticky ends on the fragment are complementary to those on the vector and bind using the base-pairing rule

Front 24

What is step 4 of insertion?

Back 24

DNA ligase is added, joining the fragments by reforming the sugar-phosphate backbone where it was broken by the restriction endonuclease. This is ligation

Front 25

What is step 5 of insertion?

Back 25

This forms a loop of recombinant DNA

Front 26

What could also happen during insertion?

Back 26

The plasmid sticky ends may reattach without taking up the DNA or the DNA could bind to itself forming a plasmid which isn't recombinant

Front 27

What is transformation?

Back 27

This is where DNA is transformed into suitable host cells

Front 28

How do we transform cells?

Back 28

We place bacterial cells and the plasmids into an ice-cold calcium chloride solution, making the bacterial cell's plasma membranes more permeable. The mixture is heat shocked by heating to 42 degrees for 1-2 mins. This encourages the cells to take up the plasmids

Front 29

Why do we need to identify the transformed cells?

Back 29

Only a small number take up the plasmids.

Some plasmids close up without incorporating the DNA.

The DNA fragment ends may join to form their own plasmid

Front 30

What do we use to identify bacteria that have taken up the plasmid and what are examples?

Back 30

Marker genes e.g. antibiotic resistance marker genes, fluorescence marker genes and enzyme marker genes

Front 31

Which antibiotics are the genes usually resistant to?

Back 31

Ampicillin and tetracycline

Front 32

What is the method of identification used with antibiotic resistant marker genes?

Back 32

Replica plating

Front 33

What happens during the insertion of the gene with antibiotic resistant marker genes?

Back 33

The restriction endonuclease cuts through the gene for tetracycline resistance nd this is where host DNA is inserted, so the bacterium is no longer resistant to tetracycline.

Front 34

What is done for identification after transformation?

Back 34

Colonies are grown on agar plates

Front 35

What three types of colonies will from and what can kill them?

Back 35

Bacteria without plasmid-killed by both

Bacteria with plasmid without the gene -killed by neither

Transformed cells- only killed by tetracycline

Front 36

What is done to the colonies after they've grown?

Back 36

They're placed on an agar plate of ampicillin using a sterile velvet pad. The plate is then left to incubate and form colonies. Bacteria without plasmids will be killed

Front 37

What is done next?

Back 37

The colonies are then transferred onto another agar plate with tetracycline using a sterile velvet pad. Only untransformed colonies survive

Front 38

What is the final step for identification?

Back 38

The colonies on the 2 plates can be compared to identify the colonies of transformed bacteria

Front 39

What type of cloning is this?

Back 39

In vivo

Front 40

What are advantages of in vivo cloning?

Back 40

The vectors can be used in gene therapy- they could transfer the gene into the cells of another organism.

There's a low contamination risk

It's very accurate

It cuts out specific genes

Transformed bacteria can produce lots of product

Front 41

What is amplification?

Back 41

Making many copies of a DNA fragment

Front 42

What are the 2 methods for amplification?

Back 42

In vivo cloning- gene copies made within living organisms.

In vitro cloning- gene copies are made outside of a living organism

Front 43

What must be present in the vector before in vivo cloning can occur?

Back 43

The vector must contain a specific promoter and terminator region before and after the gene. A promoter region is where RN polymerase can bind and a terminator region is where it unbinds

Front 44

Which method of amplification is used for in vitro cloning?

Back 44

The polymerase chain reaction (PCR)

Front 45

What is required for PCR?

Back 45

The required DNA fragment,

Taq polymerase,

Primers,

DNA nucleotides,

Thermocycler

Front 46

What is taq polymerase?

Back 46

A type of DNA polymerase which is thermostable as it's obtained from bacteria living in hot springs.

Front 47

What are primers?

Back 47

Short sequences of nucleotides that have a set of bases complementary to those at one end of each of the 2 DNA fragments

Front 48

What is a thermocycler?

Back 48

A computer controlled machine that varies temperature precisely over a period of time

Front 49

What is stage 1 of PCR?

Back 49

All of the components are added to the thermocycler and the temperature is increased to 95 degrees. This causes hydrogen bonds to break, separating the strands of the DNA fragment

Front 50

What is stage 2 of PCR?

Back 50

The mixture is cooled to 55 degrees, causing primers to anneal (bind) to their complementary bases at the end of the fragment. This allows taq polymerase to bind and start copying the DNA. It also prevents the strands from rejoining

Front 51

What is stage 3 of PCR?

Back 51

The temperature is increased to 72 degrees, the optimum temp for taq polymerase to add complementary nucleotides along each of the separate strands

Front 52

What are 2 advantages of in vitro cloning?

Back 52

It's extremely rapid

Living cells aren't required- no complex culturing required