Aim: to introduce DNA technology techniquesand its applications Objectives: To describe DNA extraction To explain and demonstrate DNA cloning To explain the process of PCR and its uses. To explain DNA fingerprinting and its uses
The structure of nucleic acidsThere are two types of nucleic acids: 1. Deoxyribonucleic acid (DNA) 2. Ribonucleic acid (RNA) Both are polymers made up of sub-units called nucleotides
Each nucleotide is made up of three parts: A Phosphate group A pentose sugar (either ribose or deoxyribose) A base which contains nitrogen
Restriction enzymes Restriction enzymes are enzymes that cut DNA into small fragments. This allows individual genes to be isolated. Restrictions enzymes recognize and cleave at specific DNA sequences. E.g. EcoRI
Vectors Vector - A DNA molecule that carries the foreign DNA fragment into a host cellSeveral types of vectors are used including:• Plasmids• Bateriophages (i.e. the DNA of the bacteriophage genome)• Viruses
Plasmids vectorsEssential features of a plasmidvector:• A origin of replication• A selectable marker that allows cells that carry the plasmid to be distinguished from cells that do not. This is usually an antibiotic resistance gene.• One or more unique restriction enzyme sites into which DNA can be ligated.
Hosts Host cells include: E. coli Other types of bacterium Yeast
Basic steps in genetic engineering1. Isolate the gene2. Insert it in a host using a vector3. Produce as many copies of the host as possible4. Separate and purify the product of the gene
Step 1: Isolating the gene • The action of a restriction enzyme
Step 2: Inserting gene into vector• Plasmid removed from bacterial cell and are cut with the same restriction enzyme• A chunk of DNA can thus be inserted into the plasmid DNA to form a “recombinant”• DNA ligase attaches human gene to plasmid at sticky ends
Step 3: inserting vector into host The recombinant plasmids are then mixed with bacteria. Vector plasmid taken up by treated bacterium This insertion is called transformation
Step 4: Multiplication of the host cells by cloning• In these conditions, the bacteria multiply rapidly, making many copies of the human gene.
Recombinants Vs non-recombinants• The plasmids have naturally occuring genes for antibiotic resistance• An agar plate containing Ampicillin is used to allow only those cells which have taken up a suitable plasmid to survive and divide. These cells must have resistance to Ampicillin
What is PCR?• PCR involves the targeted amplification of a specific DNA sequence.• Using PCR the amount of a chosen DNA fragment can be increased 10-10 fold in 2-3 hours
Performing a PCR reactionA PCR reaction contains:1. A small amount of DNA fragment to be amplified.2. The primers which act as signals to the DNA polymerase enzyme to start copying.3. The different types of nucleotide containing the bases adenine, guanine, cytosine and thymine.4. Taq DNA polymerase
Performing a PCR reaction Strands of sample DNA separated by heating to 95oC Mixture cooled to 55oC to allow primers to bind. Mixture heated to 70oC for replication (optimum temp of DNA polymerase) Animation Video
PCR AmplificationExponential Amplification of template DNA
DNA fingerpriting DNA profiling allows individuals to be unambiguously identified. It relies on differences between the genomes of different individuals. The DNA of every human being is 99.9% the same. It is the 0.1% that makes all the difference
Short Tandem Repeats (STR) An STR is a sequence of 2-5 bp (e.g. TCAT) repeated from 1- 50 times. STRs occur at many sites in the genome of humans and other animals. The number of copies of the repeat in a particular STR varies enormously between different individuals. It is the number of times that these blocks of STRs are repeated that produces the variation in individuals.
Obtaining a DNA profile The DNA is extracted from the sample and cut into millions of small fragments using resctriction enzymes, aimed at a specific base pair sequence, called a restriction site.
DNA fragments are separated using electrophoresis. The DNA samples to be analysed are each added to a well. The fragments are then subjected to an electric field The smaller fragments move faster, the larger ones move slower
The DNA fragments are transfered to a nylon membrane by a process called Southern blotting. Radioactive probes are used to attach to specific parts of the fragments. The nylon sheet with DNA fragments attached is placed under X-ray film. Patterns of bands on DNA profile
This produces a visiblepattern of light and darkbands (where the radioactiveprobe is present) rather like abar-code. Everyone’s bar-code is different
DNA fingerprinting in Forensics DNA fingerprints can be used as biological evidence Strands of DNA can be found on hair, blood or semen Useful in solving crimes like murder and rape DNA Fingerprinting has exonorated people who were falsely convicted
DNA profiling in forensic science DNA from a victim (e.g. in a blood spot) may be left on the clothing of an attacker. Or attacker may leave their DNA on a crime victim (e.g. in blood, hair or semen). In this case the DNA profile from the attacker is compared to that of suspects. A large database of DNA profiles of known offenders has been assembled.
In paternity testingFor each STR a child inherits oneallele (band on gel) from the mother,one from the father. In the example shown on the right, which shown the inheritance of a single STR, who is the father of the child?
Detection of pathogens by PCR Infections by certain pathogens can be detected by PCR using primers that recognise the pathogen genome. E.g. rabies can be diagnosed by amplifying rival nucleic acid present in viruses in saliva. • PCR is also valuable in the diagnosis of HIV – it can detect infection very soon after exposure and also newborn babies