Manipulating Genomes 6.3 Flashcards
Describe Fred Sangers’ DNA sequencing approach?
- A single strand of DNA is used as a template and placed four dishes each containg the bases A,T,C and G plus the enzyme DNA polymerase
- In each dish is a modified version of a DNA base (each dish either has A,T,C, or G of this modified base), when synthesised to the complementary strand no more bases can be added
- Thousands of DNA fragments are then generated in the dishes of varying lengths, they are then passed through a gel electrophoresis and get sorted by length
4.This is then used to work out the DNA sequence
How is DNA cloned for sequencing?
1.The gene is isolated using restriction enzymes
2. DNA is then inserted into a bacterial plasmid and cultered, dividing many times
3. Each new bacterium contains a copy of the gene and the genes can now be isolated using plasmid preparation techniques
Describe the process of pyrosequencing?
- A section of DNA is cut into fragments and split into single strands
- PCR is used to amplify the DNA fragments then they are put into seperate wells
3.Free nucleotides attach to the wells and DNA strands via complementary base pairing - Wells contain specific enzymes which cause light to be emitted when bases are added to the strands
- Computers analyse the occurence and intensities of light emitted and process the information to interpret the DNA sequence
What is bioinformatics?
Bioinformatics is the field of studying the vast quantities of data generated in biology using appropriate technological tools.
What are the applications of gene sequencing?
-Comparisons between species: comparing genomes of species can help us understand evolutionary relationships
-Epigenetics: methylation of certain chemical groups can help scientists understand the development of certain diseases
-Scientists can use gene codes to determine the sequence of amino acids in the protein it codes for
-Synthetic biology: creating new biological parts e.g genetically engineering drugs
Outline the procedure of DNA profiling?
- DNA is obtained from the individual e.g mouth swab
2.DNA is digested with restriction enzymes and cut into fragments - Fragments are seperated by gel electrophoresis, larger fragments travel a shorter distance
- A banding pattern can be seen
- The DNA for which it is being compared to goes through the same process and they are then compared
What type of DNA is usually analysed for DNA profiling?
-Short tandem repeats (STR) sequences of DNA are used
-The exact number of STRs vary from person to person
-STRs are polymorphic (contains two or more variants) so the chances of two people randomly sharing the same gene loci is very low
What are the applications of DNA profiling?
-Forensic science: has helped bring convictions and prove innocence of crime
-Maternity and paternity disputes: half of the STR fragments come from the mother and father so by comparing DNA profiles you can determine maternity/paternity
-Analysis of disease: electrophoresis of blood can for e.g detect type of haemoglobin present in sickle cell anaeimia
What is PCR?
-Polymerase chain reaction: a biomedical process thay amplifies DNA allowing it to be analysed.
-Only short sequences of 10,000 base pair can be replicated into thousands of millions of copies
Why was the PCR process initially time consuming?
-DNA had to be heated to denature it and then cooled down in order for the primers and DNA polymerase to work
-Now DNA polymerase has been obtained from thermophilic bacteria so it is able to work at high temperatures
Outline the PCR process?
1.Sample of DNA is mixed with nucleotides, primers, magnesium ions and thermophilic DNA polymerase
2. It is heated to around 96℃ to break the hydrogen bonds and denature the double stranded DNA into two single strands
3. Mixture is cooled to around 68℃ so that primers can anneal (bind) and thermophilic DNA polymerase can now bind
4. Temperature is then raised to optimum temperature for DNA polymerase enzyme
5. The DNA polymerase catalyses the addition of DNA nucleotides on the single strand in the 5’ to 3’ direction
6. A new double strand of DNA is created and the cycle is repeated, the amount of DNA increases exponentially
Define a thermophile?
-Thermophiles are heat loving organisms, they are a type of extremophile
What are the applications of PCR?
-As it is used to amplify DNA it has lots of uses involving DNA sequencing for example:
1. Forensic science
2. Reasearch of old DNA
3. Detecting mutations
What is electrophoresis?
Electrophoresis: process used to seperate proteins or DNA fragments of different sizes
What is the role of the magnesium ions in PCR
-Acts as a Cofactor for the DNA polymerase
What is a DNA probe and how is it useful?
-A DNA probe is a short single stranded length of DNA complementary to a section of DNA being investigated
-The probe can be labelled with a radioactive marker and a fluorescent marker
-They are useful for locating specific DNA sequences e.g in genetic engineering
How are ladders used in electrophoresis?
-Ladders are a standard reference that contains known lengths of DNA fragments and results of electrophoresis can be compared to find out the sizes of the fragments
What is electrophoresis and how do you carry it out?
-It is a technique used to seperate different lengths of DNA and proteins
1.Set up apparatus as shown
2. The gel is agarose molecules held together by hydrogen bonds and form tiny pores
3. The main body of the box is filled with a buffer solution that conducts current and DNA is transferred into the wells
4. Power is turned on and DNA molecules move through the gel, the smaller the fragment the quicker it moves
What are DNA microarrays?
-A number of different probes (labelled with fluorescent markers) on a fixed surface, DNA under investigation is then added and it can reveal the prescence of mutated alleles
-As the probes are made complementary to the mutated alleles
What and why is genetic engineering otherwise known as and outline simply the stages of genetic engineering?
-Genetic engineering is otherwise known as recombinant DNA technology because it involves combining DNA from different organisms
-The simple stages of genetic engineering are:
1. Required gene is obtained
2. Copy of gene is placed in vector
3. Vector carries gene into cell
4. The cell expresses the gene
What are different ways you can get the vector into the recipient cell in genetic engineering?
-Heat shock treatment: exposing cells to alternating periods of cold and heat so membranes will become more porous
-Electroporation: a high voltage pulse is applied to cell to disrupt the membrane
-Electrofusion: electrical fields help introduce DNA into cells
What are sticky and blunt ends?
-Sticky end: when the cut DNA has exposed unpaired nucleotide bases
-Blunt end: when the cut is not staggered leaving no unpaired nucleotide bases
How are DNA ligase enzymes used in molecular biology?
-It is used to join DNA fragments as it catalyses the condensation reaction that joins sugar and phosphate groups in the DNA backbone
What is the name of the restriction enzymes in archaea and bacteria?
Restriction endonculeases
What is reverse transcriptase?
-The enzyme that converts RNA into DNA, mRNA is used as a template to produce a single strand of complementary DNA
How is bacteria modified in the laboratory to make sure it can’t get out into the wild?
-They have a gene knocked out meaning they can’t synthesis a particular nutrient, they can therefore only survive in the growth medium as it contains this nutrient
What are the potential benefits and risks associated with genetic modification?
Benefits: nutritional value of food can be improved, better yield so can improve food supply in poor countries
Risks: adverse health problems, cross pollination could lead to ‘super weeds’
What is the basic principle of gene therapy?
-Is to insert a functional allele of a gene into a cell with mutated or non-functional alleles of the gene, if the inserted allele is expressed then the individual will no longer have symptoms associated with the genetic disorder
What are the different methods for delivering the functioning alleles to the patients cells in somatic cell gene therapy?
-Liposomes: alleles are packaged into small spheres of lipid bilayers called liposomes, these can for e.g be placed into inhalers and sprayed into the respiratory tract and tacken up by cells lining the tract
-Viruses: viruses can be GM to have the functioning allele and made unable to cause disease, the virus then enters the recipient cells taking the allele with it
Will a patient treated with Somatic gene therapy pass the functioning alleles onto their offspring?
No
What is germ line gene therapy?
-Involves altering the genome of gametes or zygote, so all the cells of the individual will be altered to have the functioning allele
What are the problems with germ line gene therapy?
-Zygote can not consent to having genetic makeup changed
-Could disrupt the regulation of other genes and for e.g increase the risk of cancer for the individual
