manipulating genomes 6.3 Flashcards
the principles of DNA profiling
- every person (apart from identical twins) has repeating short non-coding regions of DNA (20 to 50 bases) that are unique to them ( variable number tandem repeats VNTR). they are Introns
-The number of VNTR regions are inherited from your biological parents. The more closely related you are to a person the more likely the repeats have similar patterns
-The length of the VNTR regions are unique to each individual (apart from identical twins)
When DNA testing occurs in forensic medicine and criminal investigations the image of these repeats in the DNA (indicated by a pattern of bars) creates a DNA profile or fingerprint. The profile is analysed to allow conclusions to be made
-also can be uses in detection of diseases e.g. sickle cell anaemia or huntingtions disease by detecting the haemoglobin a person has.
procedure of DNA profiling
- Obtain the DNA
-amplify the quantity of DNA using a PCR
-Use restriction endonucleases to cut the amplified DNA into fragments
-Separate the fragments using gel electrophoresis
-use southern blotting paper to transfer the fragments on to
-Add radioactive or fluorescent probes that are complementary and therefore bind to specific VNTR regions
-X-ray images are produced or UV light is used to produce images of the fluorescent labels glowing.
the principles of genetic engineering
- the required gene is isolated and placed into a desired vector forming recombinant DNA (DNA joined together from different sources)
procedure of genetic engineering
1) DNA is isolated
2) DNA is copied during PCR
3) the DNA is cut using restriction endonuclease. The same enzyme is used to cut the plasmid of the vector therefore the sticky ends of the DNA fragment and vector our complementary
4)the vector and the DNA ligase forms phosphodister bonds between the desired gene and pasmid forming recombinant DNA. Antibiotic resistant marker are also inserted
-plasmid is inserted into the host cell. The cell membrane needs to be made more permeable for this to occur.
method:
-electroporation= a electrical shock is applied to the cell to disrupt the membrane
- the transgenic bacteria is mass produced an placed on a agar plate containing nutrients
-successful transgenic DNA can be seen using a UV light if a fluorescent marker is used or if using an antibiotic resistant gene see which antibiotics have survived. Once the antibiotics has been applied
ethical issues about engineering
positive
-soyabean plants has been modified to be insect resistant by inserting with Bt toxin
- bacteria genes modified to produce insulin
negatives
-ethical concerns : Biotech companies charge farmers more money for GM seeds vs non-GM seeds to try and make back the money they have invested in their product. Seeds can not be kept from GM crops to regrow the crop the following year because GM crops do not “breed true”
.Buying seeds year upon year can be a major struggle for farmers in developing countries
-lack of long term research into the effects of humans
The principles of DNA sequencing
- Dna sequencing is a method to isolate and read genes and deduce the order of bases
1)add to 4 different test tubes - lots of DNA
-lots of DNA primer
-DNA polymerases - Free nucleotides
- Fluorescently labelled modified nucleotides
2) heat the test tube to 95 degrees to denature the DNA separating the double helix to obtain a single strand of DNA
3)Cool to 50 degrees to allow the primers( short fragments of single-stranded DNA) to anneal and then nucleotides are added to the strand of DNA
4) heat to 60 degrees for DNA polymerase to join the nucleotides on to the strand reforming hydrogen and phospodiester bonds.
5)The flurosecently- labelled modified nucleotides is added at different points which terminates the strand producing different strand lengths.
6) the DNA fragments in each tube are separated by electrophoresis and visualised under UV which can be read from gel - smallest nucleotide = one base is at the bottom of the gel= first base and each band after represents one more base added to build up the sequence
The development of new DNA sequencing techniques
-nowadays an automated process is developed
-high-throughput sequencing - techniques that can sertence a lot aster than original methods:
- or prosequencing
-massive parellel sequencing
Use of genetic sequencing
-the human genome can be compared with those of other species.e.g found humans are 95% similar to chimpanzees
- Bioformatics= developing and using computer software’s that can analyse,organise and store
biological data
Once a genome is sequenced, bioinformatics allows scientists to make comparisons with the genomes of other organisms using the many databases available
This can help to find the degree of similarity between organisms which then gives an indication of how closely related the organisms are
- can help confirm evolutionary relationshipss.
-The genetic code can be used to predict the amino acid sequence within a protein
Once scientists know the amino acid sequence they can predict how the new protein will fold into its tertiary structure
This information can be used for a range of applications, such as in synthetic biology.
-Genotype-phenotype relationships are explored by “knocking out” different genes (stopping their expression) and observing the effect it has on the phenotype of an organism
When an organism’s genome sequence is known, scientists can target specific base sequences to knock out
-The genomes of pathogens can be sequenced and analysed to aid research and disease control( epidemiology)
Principles and application of PCR
- can be use to amplify fragments of DNA
1) A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase.
• Primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want.
2) The DNA mixture is heated to 95 °C to break the hydrogen bonds between the two strands of DNA.
3) The mixture is then cooled to between
50 and 65 °C so that the primers can bind (anneal) to the strands.
4) The reaction mixture is heated to
72 °C, so DNA polymerase can work.
5) The DNA polymerase lines up free DNA nucleotides alongside each template strand, Complementary base pairing means new complementary strands are formed.
6) Two new copies of the fragment of DNA are formed and one cycle of PCR is complete.
7) The cycle starts again, with the mixture being heated to 95 °C and this time all four strands (two original and two new) are used as templates.
8) Each PCR cycle doubles the amount of DNA.
Electrophoresis
- Use restriction enzymes to cut the DNA sample.
- Load into wells near the cathode( negatively charge electrode) of the agarose gel, along with buffer. And loading dye so the fragments sink to the bottom
- Apply an electric current and allow the fragments to separate. Stop the current before the DNA reaches the end of the gel.
- smaller fragments travel faster so further
- seperates DNA based on mass
How to see DNA fragments
-Southern blotting. using radioactive
DNA probes and x rays
-Using a GFP (green (fluorescent protein) DNA probe and UV light
Bioformatic uses
- information is universal
-Access to large amount of data on DNA and proteins;
_ Computer modelling of new protein structure from base sequence;
Use of bioformatics in epidemiology
-lIdentify the source of the outbreak; -identify vulnerable populations;
-design vaccination programs to target certain individuals;
What is recombinant DNA
-DNA combined from 2 sources
Plant vectors
-plasmids, viruses
