Biotechnology and gene technologies

Understanding and manipulating DNA

• DNA profiling is used in forensic crime scenes and paternity testing
• Genomic sequencing and comparative genome mapping is used to research function of genes and regulatory DNA sequences
• genetic engineering is used to make pharmaceutical chemicals and GM organisms
• gene therapy is ised to treat conditions such as CF.

Techniques used involved have their basis in natural processes -

• DNA strangd cut into small fragments using restriction endonuclease enzymes
• The fragments can be seperated by size in electrophoresis and then multiplied by polymerase chain reaction
• DNA fragments can be analysed to give base sequences and sealed using ligase enzyme
• DNA probes can be used to locate a specific sequence of DNA

The Genomic Age - The DNA of organisms hold sections called genes. The coding DNA for the proteins is only a small part of the entire DNA of that organism. Alot of DNA is non coding DNA; has regulatory functions.

Sequencing the genome of an organism - an outiline - The sequencing reaction can only operate on a legnth of DNA 750 base pairs long. This means the genome must be split into sections. Sequencing is carried out a number of times on overlapping fragments, which are put back to gether to form the original code.

• Genome is mapped to identify which part of the chromosome they have come from. Info from microsatelites are used as they are already known.
• Samples of the gene are sheared into smaller sections of 100,000 base pairs.
• These sections are put into bacterial artificial chromosomes (BAC) and put into E.Coli cells so many copies (clone libraries) are made.                                  

Comparing Genomes - comparative gene mapping is when you compare the genomes of organisms.

• Comparing genes for proteins gives clues to their relative importance of life.
• Comparing their DNA allows predictions of evolutionary relationships; common ancestors and how closely they are related.
• Modeling effects of changes to DNA/genes can be carried out; such as yeast. All characteristics are present in the phenotype.
• Comparing genomes from pathogenic and similar non pathogenic organism can show the base pair sequence responsible for that disease. This can lead to specific target drugs.
• DNA of individuals can be analysed and can reveal mutant alleles, or ones associated with diseases.

Electrophoresis - seperates DNA fragments based on their size.

A gel plate is used which contains agrose and a buffer solution. Electrodes are attached at either end so a current can be passed through. Seperation occurs as the longer strands get caught up in the gel and are slowed whereas the shorted ones move quickly through travelling further.

• DNA samples are treated with restriction enzyme to cut them into smaller fragments.
• DNA samples put into wells at negative end of the electrode
• Gel is put into buffer solution and electical current is put through
• DNA is negatively charged due to the phosphyl groups and is attracted to the positive electrode. DNA diffuses through gel to get to the positive end.
• Shorter strands move fast. Longer strands move slower.
• Position of fragment can be seen by a dye used in the DNA molecules.

The fragments may be further analysed by Southern blotting; nylon sheet left over gel all night and the DNA fragments diffuse onto the sheet amd can be analysed.                                                                                                           To veiw the DNA on the sheets, a radioactive or fluoresent marker is intoduced into the DNA molecule. This can be used to check a for a specific sequence.

DNA probes - short single stranded piece of DNA complimentary to the section of DNA being investigated. It is labelled using a

• radioactive marker on the phosphoryl group which shows up on photographic film
• fluorescent marker that emits a colour when exposed to UV light

The probes are added to any sample of DNA fragments and join to any fragment where complimentary base pairing occurs; annealing.                   Probes are useful for locating specific sequences because:

• locate genes wanted for genetic engineering
• identify the same gene on a variety of different genomes from separate species when comparing genomes
• to identify the presence or absence of an allele in a particular genetic sequence

Diagnoses of genetic disease and identification of symptomless carriers can be found using DNA probes. DNA micro array is when many probes are fixed and complimentary DNA anneals to this. Sample copies must be broken up.

The polymerase chain reaction - PCR; is articicial DNA replication.                      Only tiny samples of the DNA are needed, as it is multiplied; amplified.

The sequencing relies on the fact DNA:

• is made up of antiparallel backbone strands
• strands are made up of 5' prime end or 3' prime end
• grows only from the 3' prime end
• base pairs pair up according to the complementray base pairing rules

PCR is not identicle to natural DNA replication - only short strands are replicated, the addition of a primer molecule enables the reaction to start, a cycle of heating acooling is required to break and join bonds.

The DNA polymerase enzyme is described as 'thermophillic' as it is not denatured by the extreme temperatures used in the proccess. It came from thermophillic bacteria that lived at 90 degrees.

PCR is a cyclic reaction -

• The DNA is mixed with nucleotides and DNA polymerase
• The mixture is heated to 95C. This breaks the hydrogen bonds between the complementary strands together; samples are single stranded.
• short lengths of single stranded DNA are added; primers
• Temperature reduced to 55C allowing primers to bind to form small sections of double stranded DNA at each end of the sample using DNA polymerase.
• Temperature is then raised to 72C (optimum temp for DNA polymerase) and the enzyme extends the double stranded section by adding free nucleotides
• The whole process can be repeated; exponentially.

Primers -  are short single stranded sequences of DNA around 10-20 base pairs long, They are needed in the sequencing reaction and polymerase reactions to bind a section of DNA because DNA polymerase cannot bind to single stranded DNA fragments.  

Automated DNA sequencing is based on interrupted PCR and electrophoresis -

Automated sequencing is much faster than using radioactive labels. The reaction mixture as with PCR contains: DNA polymerase, single stranded template DNA fragment, free DNA nucleotides and primers. However, some nucleotides contain a fluorescence marker. If they are added to the growing chain, the strand cannot continue.

• Primer anneals at the 3' end of the template strand allowing DNA polymerase to attach.
• DNA polymerase adds free nucleotides so the strand grows; same as PCR and natural DNA replication. 
• If a modified nucleotide is added, the reaction stops on that template strand.
• As the reaction proceeds many molecules of DNA are made, The fragments vary in size.
• As these strands run through the machine a laser reads out the colour sequece (from thedifferent colours of fluorescent marking) in order of one nucleotide added, two, three etc. The sequence of colours and so the sequence of bases can be displayed.  

Genetic engineering is also called recombinant DNA technology.

Required gene is obtained - Copy of the gene is placed into a vector - The vector carries the gene to the recipient cell - Recipient expresses the gene through protein synthesis.

• Obtaining the gene to be engineered - mRNA produced from transcription can be obtained from the cells where the gene is expressed / gene synthesised using automated polynucleotide sequencer / DNA probe can be used to locate gene on DNA fragments and gene can be cut using restriction enzymes.
• Placing the gene in a vector - gene can be sealed into bacterial plasmid using DNA ligase / genes sealed into virus genomes or yeast cell chromosomes / vectors contain regulatory sequences of DNA so inserted gene is transcribed in the host cell.
• Getting the gene into the recipient cell - the gene can be large so not easily crossed between the membranes is recipient cell. Electroporation - pulses disrupt membrane. Microinjection - DNA injected using micropipette into nucleus. Viral transfer - virus' mechanism infects the DNA directly. Ti plasmids - vectors inserted into plant genome. Lipsomes - DNA wrap to liosomes and can pass through cell membrane.

Restriction enzymes cut DNA backbones; ligase enzymes seals them

• Restriction enzymes are used to cut through DNA at specific points.
• These enzymes were taken originally from bacterial cells to fight against viruses.
• A restriction enzyme will cut DNA where ever a specific base sequence occurs; restriction site.
• It catalyses the hydrolysis of the phosphate-sugar backbone.
• It leaves exposed bases; sticky ends.
• DNA ligase is used to catalyse the condensation reaction which joins the phosphate-sugar backbones of the double helix together.
• For joining to occur, fragments from different sources need to have been cut by the same restriction enzyme.
• These meant the sticky ends are complimentary and allow base pairing and hydrogen bonds to form.
• DNA ligase then seals the backbone.
• Where DNA fragments from different organisms are joined this way, the resulting DNA is called recombinant DNA.