recombinant gene technology

similarities and differences between probes and primers

similarities:
- both short, single stranded DNA sequences
- complementary to DNA target sequence of interest

differences:
- a probe has a label (fluorescent/radioactive), and a primer doesn’t have a label
- primers can be radioactive - and in this case will act as a probe
- a probe is used to identify presence of DNA fragment / gene, and a primer is used to start DNA synthesis by acting as a binding site for DNA polymerase

polymerase chain reaction (PCR)

method of copying fragments of DNA in vitro

PCR requires:
- the DNA fragments to be copied
- DNA polymerase (thermostable enzyme)
- primers (short nucleotide sequence complementary to one end of each DNA fragment strand)
- nucleotides (A,G,C,T)
- thermocycler (can precisely vary temp)

what does the PCR require?

- the DNA fragments to be copied
- DNA polymerase (thermostable enzyme)
- primers (short nucleotide sequence complementary to one end of each DNA fragment strand)
- nucleotides (A,G,C,T)
- thermocycler (can precisely vary temp)

the PCR can be used to produce large quantities of DNA. describe how the PCR is carried out.

1. DNA heated to 95 degrees Celsius, breaks hydrogen bonds and separates DNA strands

2. cooled to 55 degrees Celsius

3. annealing (binding) of primers by hydrogen bonds (primers are needed for DNA polymerase to attach and extend from)

4. nucleotides attach

5. heated to 72 degrees Celsius, optimum temp for DNA polymerase to synthesise new complementary strand of DNA (from 5’ to 3’), phosphodiester bond between adjacent nucleotides

6. cycle is repeated

protein production: gene transfer and cloning

1. isolation: of DNA fragment containing the gene

2. insertion: of DNA fragment into a vector

3. transformation: transfer the DNA into host cells

4. identification: (using gene makers) of host cells which have taken up the gene

5. growth/cloning: of the transformed host cells

in vivo cloning

DNA fragment, which codes for the human insulin gene, is now isolated, e.g. by using a gene machine

add a promoter region and a terminator region to DNA fragment (gene) so it can be transcribed and translated in a bacterium

since the gene doesn’t contain any introns, it can be transcribed and translated in a prokaryotic organism (e.g. a bacterium)

we can clone the DNA fragment in vivo (inside a living organism), e.g. by transferring the DNA fragments into a host cell (bacteria) using a vector (plasmid)

in vivo cloning - insertion of the isolated gene into a vector (e.g. a plasmid)

- a plasmid is a double stranded circular piece of DNA that is capable of replicating independently
- they’re useful cuz they nearly always contain antibiotic resistant genes
- it’s useful cuz it contains 2 antibiotic resistant genes, one for ampicillin and one for tetracycline
- the tetracycline resistant gene will be disrupted when a restriction enzyme cuts open the plasmid
- the gene is inserted into this position. resistance to tetracycline is lost. transformed bacteria will die if exposed to tetracycline
- the ampicillin resistant gene is used to help us to identify the bacteria which have taken up the plasmid (and have thus been ‘transformed’)

insertion of the gene into plasmids:
- restriction endonuclease can be used to cut the DNA to leave a sticky end
- cut the plasmid (and this cuts within the tetracycline resistance gene) using the same restriction endonuclease - to leave the same sticky end
- mix the DNA with the cut plasmids - the DNA is incorporated into the plasmid
- the enzyme DNA ligase permanently joins the sugar phosphate backbone of the DNA and the plasmid together using a phosphodiester bond
- (problem: some plasmids close up without taking up the DNA fragment)

in vivo cloning - transformation: transfer of plasmid vector containing the isolated DNA fragment into a suitable host (e.g. bacteria)

the plasmids (some containing the DNA fragment and some that may not) must be reintroduced into a host cell, e.g. bacteria. this process is called transformation.

the bacteria and plasmids are mixed together along with some Ca²⁺ ions.

by altering the temp (to heat shock) the bacteria become permeable and the plasmid can pass through the cell wall and cell membrane.

(problem: most bacteria don’t take up plasmids).

3 possible outcomes of transformation:
1 - no plasmid and no DNA fragment
2 - has plasmid and no DNA fragment
3 - has plasmid and has DNA fragment

in vivo cloning - identification of successfully transformed bacteria (i.e. that contain the plasmid and the DNA gene fragment)

less than 1% of bacterial cells take up any plasmids in the transformation process.

identify the bacteria containing a plasmid.

the bacteria doesn’t have any antibiotic resistance genes of their own. the antibiotic resistant genes are only found in the plasmid. so the only way the bacteria can become resistant to the antibiotics is by taking up a plasmid.

if we grow the transformed bacteria on a medium containing the antibiotic ampicillin any bacteria that survive must contain a plasmid.

gene markers: identification of bacteria with a plasmid that contains the DNA fragment

- the transformed bacteria now contain plasmids
- but only some of those plasmids contain the DNA fragment
- so we must identify a bacterium that has been transformed with a plasmid, but one that contains a plasmid with the DNA fragment.

- gene markers are used to identify which plasmids have taken up the DNA fragment
- gene markers can be used for:
→ antibiotic resistance: transformed bacteria aren’t resistant to an antibiotic
→ making a fluorescent protein: e.g. green fluorescent protein. transformed bacteria can’t fluorescence
→ making an enzyme with an identifiable product: e.g. the lactase enzyme turns a colourless substrate blue. transformed bacteria can’t turn the substrate blue, and will appear as white colonies.
- the gene marker is disrupted if the DNA fragment is present and the gene won’t be expressed

fluorescent gene markers

the gene from jellyfish produces green fluorescent protein (GFP)

if the DNA fragment has been inserted into the GFP gene, the bacteria will glow and wouldn't be seen

outcome if gene inserted: no fluorescence

outcome if no gene inserted: fluorescence

enzyme gene markers

- the enzyme lactase turns a colourless substrate into a blue colour
- if the gene has been disrupted by the incorporation of the DNA gene fragment, a functional enzyme won’t be made so the substrate will remain colourless
- outcome if gene inserted: colour of colony = white
- outcome if no gene inserted: colour of colony = blue

antibiotic-resistance gene markers

the tetracycline resistance gene is disrupted when the restriction endonuclease cuts open the plasmid and the DNA is inserted

if the DNA fragment has been inserted into the tetracycline resistance gene, the bacterium will no longer grow on medium containing tetracycline

bacteria containing plasmids with no DNA fragment will still be able to grow on a medium containing tetracycline

in order to identify bacteria with a plasmid that contains the gene / DNA fragment, we use a process called replica plating

replica plating

all bacteria grow on the master plate

only bacteria transformed with a plasmid grow on the ampicillin plate

only bacteria transformed with a plasmid, that don’t have the DNA gene fragment grow on the tetracycline plate. (we have to go back to the ampicillin plate and select colonies that: grow on ampicillin plate and not on tetracycline plate)

in vivo - growth/cloning of the successful host cells

following successful identification of the bacteria containing the plasmid and the DNA fragment, the bacteria are cloned

as the bacteria are cloned, so is the plasmid containing the DNA fragment

this type of gene cloning is in vivo (cloned within a living organism)

explain why the primers used in the PCR will bind to panda DNA, but not to DNA from bacteria or bamboo

primer has specific base sequence

what is a DNA probe?

1. short single strand of DNA
2. bases complementary with DNA

Describe how the DNA is broken down into smaller fragments.

restriction endonuclease cuts DNA at restriction site

The DNA on the nylon membrane is treated to form single strands. Explain why.

so DNA probe attaches

what is meant by a non-coding base sequence?

does not code for amino acid

describe the roles of 2 named types of enzymes used to insert DNA fragments into plasmids.

1. restriction endonuclease to cut plasmid
2. ligase joins gene to plasmid

suggest three features of the structure of different proteins that enable them to be separated by gel electrophoresis.

1. number of amino acids
2. charge
3. R groups differ

suggest one reason why DNA replication stops in the polymerase chain reaction.

limited number of primers

recombinant DNA technology

- involves the transfer of fragments of DNA from one organism, or species, to another
- since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient (transgenic) organism

fragments of DNA can be produced by several methods, including:

- conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase
- using restriction enzymes to cut a fragment containing the desired gene from DNA
- creating the gene in a ‘gene machine’

how would you use a radioactively labelled DNA probe to show that certain cells contained a specific gene?

1. extract DNA and add restriction endonucleases
2. separate fragments using electrophoresis
3. treat DNA to form single strands
4. the probe will bind to the gene
5. use autoradiography to show the bound probe