Manipulating Genomes Flashcards
Define electrophoresis
Process used to separate proteins or DNA fragments of different sizes
Describe the step by step electrophoresis
1- DNA samples are digested with restriction enzymes to cut them into fragments
2-the agrose gel is made up poured into the central region of the tank whilst combs are in place at one end .once the gel is set , buffet solution is added , the combs are then removed
3-a loading dye is added to the tubes containing the digested DNA
4- the digested DNA and loading dye is added to wells in the electrophoresis gel. To do this , a pipette is used and this is held in the buffer solution just above the wells .
5- the electrodes are turned on
6- DNA fragments move through the gel at different speeds , smaller fragments travel faster so in a fixed time travel further
7- the buffer solution is poured away and dye is added to the gel , this dye adheres to the DNA and strains the fragments
Describe separating proteins
The process for separating proteins is the same as for sequencing DNA fragments but Is often carries out in the presence of a charged detergent such as sodium dodecyl sulfate (SDS) , which equalises the surface charge on the molecules and allows the proteins to separate as they move through the gel , according to their molecular mass
Describe using DNA probs
A DNA probe is a short (50-80 nucleotides ) single stranded length of DNA that is complementary to a section of the DNA being investigated
The DNA probs may be labelled using
- a radioactive marker usually with 32p in one of the phosphate groups in the probe strand . Once the probe had bound , by complementary base pairing , to the piece of DNA, it can be revealed by exposure to photographic film
- a fluorescent marker that emits a colour on exposure to UV light . Florescent markets may also be used in automated DNA sequencing
DNA probs are useful in locating specific DNA sequences , for example
- to locate a specific gene needed for use in genetic engineering
- to identify the same gene in a variety of different genomes from different species when conducting genome comparison studies
- to identify the presence or absence of a specific allele for a particular genetic disease or that gives susceptibility to a particular condition
Separating proteins can be useful to analyse the type of haemoglobin proteins for diagnosis of conditions such as
- sickle cell anaemia , where the patient has haemoglobin S and not the normal haemoglobin A
- aplastic anaemia,thalassaemia , leukaemia
Describe Fred dangers DNA sequencing approach
- uses a single strand of DNA as a template. there are 4 dishes containing a solution with the 4 bases and DNA polymerase
- to each modified version of one of the DNA bases. the base was modified in such way that once incorporated into the synthesised complementary strand of DNA, no more bases can be added , each base is labelled with a radioactive isotope
- as the reaction progressed, thousands of DNA fragments were passed through a gel by electrophoresis . smaller fragments travel further, so the fragments so the fragments become sorted by length
Describe cloning DNA
the gene to be sequenced was isolated , using restriction enzymes , from a bacterium
the DNA was then inserted into a bacterial plasmid ( a vector ) and then into an E coli bacterium host that, when cultured divided many times, enabling the plasmid with the DNA insert to be copied many times
each new bacterium contained a copy of the candidate gene . these lengths of DNA were isolated using plasmid preparation techniques and were then sequenced
Describe the first DNA sequencing machine
fluorescent dyes instead of radioactivity were used to label the terminal bases. these dyes glowed when scanned with a laser beam , and the light signature was identified by computer . this method dispensed the need for a technician to read the autoradiograms.
Describe pyrosequencing
1- a long chain of DNA to be sequenced must be mechanically cut into fragments 300-800 base pairs, using a nebuliser
2- these lengths are then degraded into single stranded DNA . these are the template DNA and they at immobilised
3- a sequencing primer is added and the DNA is the incubated with the enzyme DNA polymerase , ATP sulfurylase , luciferase, apyrase and the substrate adenosine 5 phosphosulfate (APS) and luciferin . only one of the four possible activated nucleotides, ATP,TTP, CTP and GTP is added at any one time and any light generated is detected
4- one activated nucleotide ( a nucleotide with two extra phosphoryl group ) such as TTP , is incorporated into a complementary strand of DNA using the strand to be sequenced as a template . as this happens the two extra phosphoryls are released as pyrophosphate . in the presence of APS , the enzyme ATP sulfurylase converts the pyrophosphate to ATP . in the presence of this ATP , the enzyme luciferase converts luciferin to oxyluciferin . this conversion generates visible light which can be detected by a camera
the more light , the more nucleotides have been incorporated into the complementary DNA
unincorporated nucleotides are broken down by apyrase
Describe bioinformatics
A branch of biology to store a huge amounts of data generated by pyrosequencing
Human genome project
Describe comparisons between species
when the human genome was compared with those of other species, it become clear that few human genes are unique to us . most of our genes have counterparts in other organisms . this verifies that genes that work well tend to be conserved by evolution
sometimes as evolution progresses , some genes are co-opted to perform new tasks . tiny changes to a gene in humans called FOZP2, which is found in other mammals including mice and chimpanzees , means that in humans this gene allows us to speak
many of the differences between organisms are not because some of their
Human genome project
Describe evolutionary relationships
comparing genomes of organisms thought to be closely related species has helped confirm their evolutionary relationships or has led to new knowledge about the relationship and in some cases to certain organisms being reclassified
the DNA from bones and teeth of some extinct animals can be amplified and sequenced so that the animals evolutionary history can be verified
Human genome project
Describe variation between individuals
all humans are genetically similar . except for rare cases where a gene has been lost by deletion . we all have the same genes but different alleles
the places on the DNA where these substitutions occur are called single nucleotide polymorphism (SNP) . some have no effect on the protein , some can alter a protein or alter the way a piece of RNA regulates the expression of another gene
methylation of certain chemical groups in DNA plays a major role in regulating gene expression in eukaryotic cells. methods to map this methylation of whole human genomes can help researchers to understand the development of certain diseases
Human genome project
Describe predicting the amino acid sequence of proteins
determining the sequence of amino acids within a protein is laborious and time consuming . however , if researchers have the organisms genome sequenced and know which gene codes for a specific protein , by using knowledge of which base triplets code for which amino acids , they can determine the primary structure of proteins . the researcher needs to know which part of the gene codes for axons and introns
Describe synthetic biology
is an interdisciplinary science concerned with designing and building useful biological devices and systems . it encompasses biotechnology, evolutionary biology, molecular biology, systems biology and biophysics . its ultimate goal may be to build engineered biological systems that store and process information, provide food, maintain human health and enhance the environment
Describe microarray
Scientists can place a number of different probes on a fixed surface , known as DNA. Microarray .applying the DNA under investigation to the surface can reveal the presence of mutagenic alleles that match the fixed probes , because the sample DNA will bind to any complementary fixed probes
The sample DNA must first be broken into smaller fragments and it may also be amplified using the polymerase chain reaction .
A DNA microarray can b made with fixed probes , specific for certain sequences found in mutated alleles that cause genetic disease in the well .
Reference and test DNA samples are labelled with fluorescent markers . Where a test subject and a reference markers both bind to a particular probe, the scan reveals fluorescence of both colours indicating the presence of the particular sequence in the test DNA
Define polymerase chain reaction
A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies
The PCR is artificial replication of DNA , it relies on the facts that
- DNA is made of two anti parallel backbone strands
- each strand of DNA has a 5 end and a 3 end
- only DNA grows from the 3 end
- base pairs pair up according to complementary base pairing rules (AT, GC)
the PCR differs fro DNA replication in that
- only short sequences, of up to 10000 base pairs, of DNA can be replicated , not whole chromosomes
- it requires the addition of DNA primer molecules to make the process start
- a cycle of heating and cooling is needed to separate the DNA stands , bind primers to the strands and for DNA strands to be replicated. where as in the body we use a restriction enzymes
give some examples of synthetic biology applications
- information storage- scientists can encode vast amounts of digital information onto a single strand of synthetic DNA
- production of medicines- E coli and yeast have both been genetically engineered to produce the precursor of a good antimalarial drug
- novel proteins- designed proteins have been produced , for example one that is similar to haemoglobin and binds to oxygen , but not to carbon monoxide
describe bioethics
synthetic biology raises issues of ethics and biosecurity . extensive regulations are already in place . there are many advisory panels and many scientific papers have been written on how to manage the risks . synthetic biology is not about making synthetic life forms from scratch , but is about a potential for new systems with rewards and associated risks to be managed
describe the PCR process
1- the sample of DNA is mixed with DNA nucleotides, primers, magnesium ions and the enzyme Taq DNA polymerase
2- the mixture is heated to around 94-96% to break the hydrogen bonds between complementary nucleotide base pairs and thus denaturing the double stranded DNA into two single strands of DNA
3- the mixture is cooled so that the primers can bind by hydrogen bonding to one end of each single strand of DNA . this gives a small section of double stranded DNA at the end of each single stranded molecule
4- the Taq DNA polymerase enzyme molecules can now bind to the end where there is a double stranded DNA . taq polymerase is obtained from a bacterium that lives at high temperatures
5- the temperature is raised which keeps it single stranded
6- the Taq DNA polymerase catalyses the addition of DNA nucleotides to the single stranded DNA molecules , starting at the end with the primer and proceeding in the 5 to 3 direction
7- when the Taq DNA polymerase reaches the other end of the DNA molecule, then a new double strand of DNA has been generated
8- this whole process begins again and is repeated for many cycles
the amount of DNA increased exponentially 1-2-4-8-16-32
