Gene technologies allow the study and alteration of gene function allowing a better understanding of organism function and the design of new industrial and medical processes

Question 1

Recombinant DNA technology

Answer 1

Involves the transfer of DNA fragments from one organism or species, to another

Transferred DNA can be transcribed/translated into proteins within cells of the recipient (transgenic) organism

This is possible as the genetic code is universal, as are transcription and translation mechanisms

Question 2

Fragments of DNA can be produced by several methods

Conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase

Answer 2

1. mRNA is firstly isolated from a cell that readily synthesises the protein coded for by the desired gene
2. Mix mRNA with DNA nucleotides and reverse transcriptase; reverse transcriptase uses mRNA as a template to synthesise a single strand of cDNA
3. DNA polymerase forms a second strand of DNA (double stranded gene) using cDNA as a template

Question 3

Fragments of DNA can be produced by several methods

Using restriction enzymes to cut a fragment containing the desired gene from DNA

Answer 3

1. Different restriction endonucleases cut DNA at specific sequences of bases called a ‘recognition sequence’ The shape of the recognition site is complementary to the active site
2. Some restriction enzymes are cut in a staggered fashion which creates ‘sticky ends’ (uneven cut is left in which each strand of DNA has exposed, unpaired bases) that can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences

Question 4

Fragments of DNA can be produced by several methods

Creating the gene in a ‘gene machine’

Answer 4

More recently, a new kind of technology has been developed that synthesises fragments of DNA from scratch without the need for a pre-existing DNA template

DNA fragments produced quickly/accurately via a database

Free of introns; can be transcribed by a prokaryote that can’t remove introns

Question 5

Advantages of using mRNA to make DNA fragment rather than restriction enzymes to cut gene from DNA

Answer 5

More mRNA in cell than DNA so it is easily extracted

Introns are removed by splicing (in eukaryotes) whereas DNA contains introns

Bacteria can’t remove introns

Question 6

Promotor and terminator regions added to fragments of DNA

Answer 6

Promotor and terminator regions need to be added in order for the gene to be transcribed and then translated into a protein

Promotor regions are DNA sequences that tell RNA polymerase when to start producing mRNA

Terminator regions – tell RNA polymerase when to stop producing mRNA

Question 7

The use of restriction endonucleases and ligases to insert fragments of DNA into vectors

Answer 7

Vector transports DNA into host cell e.g. plasmids or bacteriophages

Vector DNA and DNA fragments are cut open using same restriction enzyme (endonuclease) that was used to isolate the DNA fragment containing the target gene

Vector DNA and DNA fragments have complementary sticky ends i.e. complementary base pairs

DNA ligase forms phosphodiester bond between adjacent nucleotides on sticky ends

This process is called ligation

Question 8

Transformation of host cells using the vectors

Answer 8

Host cells have to be persuaded to take in the plasmid vector and its DNA

e.g. host cells placed into an ice-cold calcium chloride solution to make cell walls more permeable, then plasmids added, and mixture is heat shocked

With a bacteriophage vector, you can infect the host bacterium by injecting its DNA into it,

The phage DNA (with the target gene in it) integrates into bacterial DNA

Question 9

The use of marker genes to detect genetically modified (GM) cells/organisms

Answer 9

Not all cells/organisms will take up the vector and be transformed

Marker genes, inserted into vectors at same time as target gene, are added in order to identify which cells have the desired gene

Gene markers can be resistance to an antibiotic, fluorescent protein or an enzyme whose action can be identified

Question 10

In Vitro amplification uses the polymerase chain reaction (PCR)

Outline

Answer 10

PCR amplifies specific DNA fragments (in every cycle, amount of DNA doubles)

The reaction mixture is made up of DNA fragment + DNA polymerase (taq polymerase) + (forward/reverse)
primers + nucleotides

The cycle is repeated 30-40 times to make lots of copies of the DNA fragment

Question 11

In Vitro amplification uses the polymerase chain reaction (PCR)

Process

Answer 11

1. Strand separation occurs at 95°c, which separates DNA strands by breaking hydrogen bonds between bases
2. Allow mixture to cool to 55°c so that primers can bind (anneal) to the DNA fragment template strand, forming hydrogen bonds.

The primer (short, single stranded DNA fragment) is complementary to template DNA at the edges of the region that is to be copied

DNA polymerase binds to primer to start synthesis; it can only add nucleotides onto pre-existing 3’ end

Two different primers required (forward and reverse) because DNA strands run in anti-parallel, but polymerase can only run in one direction

3. DNA strand synthesis occurs; the optimum temperature for DNA polymerase to make complementary copies of DNA is 72°c

Nucleotides align next to complementary exposed bases

DNA polymerase joins adjacent nucleotides, forming phosphodiester bonds

Therefore, two new copies of the fragment of DNA are formed and one cycle of PCR is complete (each PCR cycle doubles the amount of DNA)