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46 Cards in this Set
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one of the most common monogenic disorder
what gene is involved, and what does this gene involve how does it lead to diseae |
. We mentioned that cystic fibrosis was one of the most common monogenic disorders.
The gene for this disorder has been identified as one that codes for a membrane protein involved in ion transport - the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR controls the export of chloride ions in the epithelial cells of the airways in the lungs. Mutations in the CFTR gene cause failure of CFTR protein to work and this leads to a build up of sticky mucus. Bacterial pathogens invade this mucus leading to disease. Remember gene names may indicate that they are disease genes. In fact CFTR is a normal gene with an important function. It is the failure of this gene to function in CF patients that has resulted in the association of this gene with the disease. |
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2most common disorder associated with the globin gene mutations
what happens in both of these what must we be aware of with regards to the heterozygosity of one of these genes |
In last weeks lecture we also looked at inherited disorders associated with mutations in the globin genes.
The two most common are Sickle-cell anaemia and a family of disorders known as the thalassaemias. Again these are examples of autosomal recessive disorders. In both these disorders, abnormalities in haemoglobin structure are responsible for the disease characteristics. Both result mainly from single base mutations that affect how the beta globin molecules form the structural tetramers of Haemoglobin that carry oxygen around the body One important point to be aware of is that in areas where malaria is endemic heterozygosity (i.e.) carrying one defective copy of the Sickle-cell gene leads to increased resistance to malaria (results in a balanced polymorphism). In this way there is a selective pressure to maintain this otherwise harmful gene in the population. |
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what is dentinogenesis imperfecta.
what type of disease results from e.g. |
Dentinogenesis imperfecta is a disease of poorly formed dentine and results in early tooth loss. There are several autosomal recessive forms of this disorder which is quite often the result of a single base mutation.
For example one form is caused by a C to T substitution in the DSPP gene (dentin sialophosphoprotein). DSPP is located on Chromosome 4 at position 4q12-23 |
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when does a carrier manifest the disease
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It is only when a caarier member in this family produces children with another carrier that the disease manifests itself.
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amelogenesis imperfecta is what type of disease
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it is an x linked disorder disease therefore
It is only when a caarier member in this family produces children with another carrier that the disease manifests itself. |
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why are close relationship marriages discourages
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We see here that both parents are carriers of the disease gene and therefore around 50% of the children will show the disease. It is for this reason that in most societies close-relative matings such as brother-sister are not permitted as the genetic similarity between related individuals carries too great a risk of producing genetically impaired offspring
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what type of disease is huntingtons
sympotoms starts when? autosomal....therefore chances if parents have it? molecular basis is what contrast with normal people severity of sympotoms |
We have previously mentioned Huntingdons disease as an example of an autosomal dominant disorder.
Sometimes referred to as Huntingdons chorea this disorder causes depression, dementia and involuntary spasms (chorea). It usually starts in middle age and as it is autosomal dominant it means that if one of your parents has it you have 50% chance of having it too. The molecular basis of the disease is an increase in the number of repeats of the triplet sequence CAG in a gene linked to the disease. Normal people have around 15 copies of this repeat sequence, HD sufferers have 36 or more. It has been shown that the more copies of this repeat that you have in your genetic make-up, the more severe the symptoms are. |
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why an an autosomal dominant disorder devastate a family
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the pedigree for a family with an autosomal dominant disorder it really does devastate the family. As there is no need for two copies of the gene for the disease to manifest itself then every individual who receives just one copy of the gene will show the symptoms associated with that disorder
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another disorder associated with blood
symptoms enamel? sympotoms, outline a phenomemon of this enamel disease explain lyonisation |
Another disorder associated with blood is Haemophilia which results in an inability to form blood clot to prevent bleeding.
. In dentistry one of the most common sex-linked anomalies is Amelogenesis imperfecta which results in poorly formed tooth enamel. The mutation is often associated with the gene for the tooth enamel protein amelogenin which is located on the X chromosome. One phenomenon associated with this disorder is the appearance of striped teeth in carrier females. This results from the process known as Lyonisation in which one copy of the X chromosome in each cell in the female body is inactivated. The inactivation is random meaning that any cell may have a functional X chromosome from the father or from the mother but not both. In the case of enamel formation this results in teeth were there are stripes of normal enamel separated by stripes where there is no enamel, due to the faulty gene being expressed |
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explain how with amelogenesis imperfecta, there can be no transmission from father to the son
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It is often possible to suggest an X-linked aetiology of disease by studying family pedigrees. In this family we see that there are only female carriers and only male sufferers. There is also no evidence of father to son transmission of the condition. This is because the disorder is on the X-chromosome and male children cannot receive an X chromosome from there fathers. They must inherit the Y chromosome in order to be male.
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explain multifactorial diseases
why may/may not manifest themselves |
Many disorders for which, there was no evidence of genetic involvement are now known to have a genetic basis. The reason why it has been difficult to establish these as genetic disorders is that for each of them there may be many genes involved. Therefore it is possible to inherit several genes which may be associated with susceptibility to a disease and still not manifest the disease as other key genes are absent.
Furthermore it is possible to have a set of genes that may be expected to result in disease but still not manifest the disease. This can be the result of another gene being present that modifies the way the genes are expressed and may be protective. |
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what is difficult to determine with multifactorial diseases,
outline several types of multifactoral diseases |
With this type of disorder it is often very difficult to determine the underlying genetic components of the disease. There are many types of multifactorial diseases and several important ones are shown in this table.
epilepsy, cleft lip and palate alzheimers schizophrenia |
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name given to disorder affected by multiple genese
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polygenic disorders
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multifactorial diseases can be influenceed how
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With this type of disorder it is often very difficult to determine the underlying genetic components of the disease. There are many types of multifactorial diseases and several important ones are shown in this table.
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what has become evident with periodontal disease
in some individuals |
some individuals even with excellent oral health still manifested the disease
It has become evident that there are other factors involved With some individuals an altered response to infection increases the severity of the disease in a manner similar to autoimmune disorders were the bodies own defences turn against themselves and start to destroy normal healthy tissue. In some of these individuals the increased tissue destruction associated with periodontal disease results from genes which act as genetic modifiers. |
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explain how different indiividuals may elitcit a different response upon exposure to the same disease initiation factors
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the presence of pathogenic oral microorganisms provides the stimulus for development of periodontal disease. Here we see three individuals with different genetic backgrounds and each responds differently to the presence of the pathogens. We can see that the disease severity is much reduced in individual A compared to individuals B and C. This difference can be a result of the presence of protective alleles in patient A compared with susceptibility genes in individual C.
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what method have we used previously to determine a genetic component of a gene
other methods that can be used |
We have previously shown that we can use family studies – looking at the number of people in a family who share the same “disorder”- to determine whether a disorder has a genetic component. Moreover, twin studies comparing monozygotic (identical) with dizygotic (non-identical) twins has proved particularly useful in dissecting out the components of a disease that are considered to be genetic and which are environmentally determined (i.e. nature versus nurture). These approaches can be informative but they cannot tell us what the underlying genetic defect is at the molecular level. In order to do this we need to establish laboratory procedures for looking at genes.
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One method of determining which chromosome an inherited defect resideds on is linkage analysis, this involves
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One method of determining which chromosome an inherited defect resideds on is linkage analysis, this involves following the inheritance of a number of known genetic markers in a n extended family pedigree to find those that appear to be present in all the affected family members.
Once a chromosomal location has been assigned it is possible to use more refined genetic tools to try to establish the nature of the genetic “lesion” involved. Today I want to briefly outline the basis of some of these techniques and explain how they can be used. We are chiefly gong to examine gene cloning, nucleic acid blotting and hybridisation approaches, the polymerase chain reaction and DNA sequencing Much of these advances have relied on constantly improving computer technology, both hardware and software to facilitate the analysis of entire genomes. Recent advances such as microarrays and “gene chips” will be covered later in the course. |
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MOST IMPORTAN PIECE OF BIOLIGICAL RESEARCH IN RECENT YEARS HAS BEEN?
WHAT HAS THIS ALLOWED US TO DO |
Perhaps the most important piece of biological research in recent years has been the human genome project. This project has resulted in the determination of the entire nucleotide sequence of the human genome. That is the entire genetic make-up of a human. Armed with this information it has been possible to identify all the genes that go to make up a human being. By comparing the “normal sequence” with those obtained for genes in individuals with genetic disorders it has been possible to begin the elucidation of the basis of specific genetic disorders. Finding these genes will allow for development of accurate diagnostics and perhaps in the longer term for treatment of those diseases
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WHY IS THE SEQUENCING PROJECT SUCH A TASK
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HUMAN GENOME IS 3 BILLION BASES WHICH IS MUCH LARGER THAN MOST OTHER ORGANISMS
THIS REQUIRED ADVANCES IN TECHNOLOGY AND COMPUTER DEVELOPMENT |
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WHAT REVOLUTIONISED MOLECULAR GENETIC ANALYSIS
WHAT DID THESE DO |
The discovery of enzymes that can recognise specific sequences in DNA and cut the DNA strands at, or near to, these sequences revolutionised molecular genetic analysis. The most useful of these enzymes recognise a 4-6bp sequence of DNA and cleave the phosphodiester bond at that point. There are many such enzymes, usually derived from bacteria and are named from the bacterial species that they are derived from. e.g. EcoRI is the first enzyme to be isolated from E. coli strain RY13
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WHAT WILL THESE RESTRICTION ENZYMES ALWAYS DO
WHAT CAN BE DONE WITH THE RESTRICTED FRAGMENTS |
As these enzymes recognise a specific sequence they will always cur a piece of DNA in the same way producing a characteristic restriction pattern of DNA fragments. Restricted fragments can be separated by size using gel electrophoresis and the sizes of the fragments generated can be estimated by analysing fragments of known size alongside samples of restriction enzyme digested DNA.
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WHAT DOES GEL ELECTROPHORESIS INVOLVE, WHY DO THE MOLECULES MOVE THE WAY THAT THEY DO
WHAT DOES IT ALLOW |
This shows an example of the patterns generated by some samples of DNA digested with the enzyme EcoRI and separated by agarose gel electrophoresis. Using this technique the fragments are separated according to size with the smallest fragments travelling furthest. All fragments migrate towards to anode as they all carry a negative charge resulting from the phosphate groups in the sugar-phosphate backbone of the DNA molecules. The agarose acts as a molecular sieve holding back the larger molecules.
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HWO DO ANTIBIOTICS BECOME RESISTANT
PLASMIDS BECAME USEFULL AS, .... FOR DOING WHAT? LEAD TO THEIR APPLICATION IN? FOR THEM TO BE USEFULL WHAT ARE THE 3 BASIC REQUIREMETNS |
The next important development was the discovery of antibiotic resistant bacteria, or more specifically the mechanism by which they became resistant. Antibiotic resistance was found to be a result of bacteria picking up and extra piece of DNA that carried antibiotic resistance genes. These Extrachromosomal DNA molecules that confer antibiotic resistance were found to be circular and were named plasmids. Plasmids became useful as vectors for carrying new pieces of genetic information and led to their application in gene cloning. For plasmids to be useful as cloning vectors there are 3 basic requirements:
1. the ability to self replicate within a host bacterium, 2. possession of a selectable marker for example antibiotic resistance and 3. They must contain useful recognition sites for restriction enzymes. |
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In order to clone a DNA fragment into a plasmid vector ....?
ROLE OF LIGASE? |
In order to clone a DNA fragment into a plasmid vector the vector and target DNA are digested with the same enzymes to generate compatible ends. The vector and insert DNA can then be joined together using DNA ligase. DNA ligase catalyses formation of phosphodiester bonds. Finally the resulting recombinant DNA can be introduced into cells treated to make them permeable
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he fragment to be cloned has been prepared using the same enzyme as
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he fragment to be cloned has been prepared using the same enzyme as was used for the vector.
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WHAT IS THEN DONE TO THE RECOMBINANT PLASMID
WHY CAN THEY BE IDENTIFIED Once a bacterial cell containing the recombinant plasmid has been identified it can be used to |
he recombinant plasmid is then mixed with a suitable bacterial host and the bacteria which have taken up the recombinant molecule are selected for by virtue of the antibiotic resistance gene on the plasmid.
Once a bacterial cell containing the recombinant plasmid has been identified it can be used to generate unlimited amounts of the recombinant plasmid and consequently the fragment of DNA cloned into it. In this way large amounts of single genes can be generated to examine their properties. |
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WHY HAS THERE BEEN A REDUCED NEED FOR RESTRICTION ENZYMES
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With the advent of the polymerase chain reaction (PCR) there has been a reduced need for restriction enzymes and DNA cloning. That is because using this technique allows the generation of seemingly unlimited amounts of a gene without the need to clone it first. PCR allows the exponential amplification of DNA molecules using DNA polymerase. Only the nucleotide sequence flanking the DNA to be amplified needs to be known for design of primers. The primers used to start the polymerase process are short oligonucleotides (~20-35) representing forward and reverse sequences (this will become clearer when we look at the diagram)
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HOW DOES PCR WORK
OUTLINE THE PROCESS |
Starting with as little as one target molecule it is possible to generate millions of copies. Firstly the target DNA is denatured by heat to make it single-stranded. The oligonucleotide primers, shown here as yellow or purple arrows are allowed to anneal to complementary DNA sequences. Once the primers have been annealed the enzyme makes a copy of each strand of the DNA by incorporating nucleotides.
he result is a doubling of the number of starting molecules. These are then in turn denatured, primers allowed to anneal and the polymerisation allowed to proceed. After the next cycle of: denaturation, annealing of primer and polymerase extension, the |
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THEORETICALLY AFTER 20 CYCLES
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Theoretically, after 20 cycles there will be more than 2 million copies of the target DNA even if we only started with one copy.
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ANOTHER USEFULL PROCEDURE FOR LOOKING AT CHANGES IN DNA
BENEFIT OF THIS TECHNIQUE OVER OTHERS |
One of the more useful procedures for looking at changes in DNA is the use of hybridisation and blotting techniques. There are many variations of this procedure but Southern blotting has proved very useful as it allows for visualisation of specific DNA fragment among thousands of molecules and can also detect gross alterations in gene structure.
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AS WE SAW IN PCR
FOR ANNEALING TO OCCUR WHAT IS NECESSARY WHAT DOES THIS ALLOW US TO DO |
As we saw when we looked at PCR, heating causes DNA to denature into single strands. They can also re-anneal when temperature lowered. For annealing to occur it is essential that two strands of DNA are complementary, as was seen with the oligonucleotide primers used for PCR. Using this important phenomenon we can search out for the presence of a specific piece of DNA or a gene target amongst thousand or even millions of other DNA molecules. We can sometimes also use this approach to detect gross changes in the structure of the target molecule.
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PRINCIPLE STEPS IN THE SOUTHERN BLOTT ANALYSIS
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This diagram outlines the principle steps in a Southern blot analysis. First the DNA is digested with a restriction enzyme and separated by agarose gel electrophoresis. The DNA fragments are then blotted onto an immobilised membrane such as nitrocellulose. This “blot” is then soaked in a buffer containing a radioactively labelled probe that is complementary to the gene of interest. After incubating the blot with the probe, the excess probe is washed off and the blot exposed to X-ray film. When the X-ray is developed, only those DNA fragments to which the probe hybridised will be visible on the X-ray film.
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IN SOME FAMILIES SICKLE CELL ANAEMIA RESULTS FROM
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In some families Sickle –cell anaemia results from a mutation which destroys one of the recognition sites fro this enzyme. This results in a single gene fragment being generated of 1350 bases in length.
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USING SOUTHERN BLOTT ANALYSIS WHAT CAN WE DETECT
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Using Southern Blot analysis we can detect the presence of this mutation in the DNA of individuals. Normal individuals will give the expected two band profile on Southern blots, giving bands of the expected sizes.
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IN ORDER TO LOOK AT GENES AT AN INDIVIDUAL LEVEL WAHT IS IMPORTANT
WHAT IS THE TECHNIQUE WHAT DOES IT RELY UPON, AND WHAT DOES THIS DO |
In order to look at genes at the single base level it is essential that we have the capabilities of determining the sequence of nucleotides in a gene. The development of the Sanger sequencing technique, by Nobel laureate Fred Sanger of Cambridge University facilitated the automation of sequencing which eventually led to the large scale sequencing projects of today. This technique relies upon a novel type of nucleotide, the dideoxynucleotide to terminate randomly, chain elongation during DNA synthesis. The process relies on four separate reactions carried out simultaneously, each containing all four dNTPs and one of the four different dideoxy nucleotides. By incorporating radioactive nucleotides into growing DNA chains it was possible to visualise the reaction by separating out the DNA fragments by electrophoresis and then exposing them to X-ray flm. strands
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DIFFERENCE BETWEEN THE ...
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The difference between a deoxynucleotide triphosphate and a dideoxynucleotide triphosphate is the absence of an oxygen atom on the 3rd carbon of the deoxyribose sugar moiety of the dideoxynucleotide. The absence of this oxygen atom means that if one of these molecules is incorporated into a growing DNA chain then no more nucleotides can be added. This is because DNA synthesis relies upon the presence of a free hydroxyl group on this third carbon atom in the deoxyribose sugar.
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HOW DOES THE SANGER TECHNIQUE QORK
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This figure outlines what a sequencing experiment will look like. The four reactions each contain a terminating dideoxynucleotide triphosphate along with all four normal bases. During the enzymic reaction some of the dideoxynucleotides will be incorporated at specific sites causing premature termination and truncated DNA fragments. When these are separated by electrophoresis they will appear as bands on the X-ray film. The sequence of the DNA can be determined by “reading” the terminated reactions from the bottom to the top as the smallest fragments run furthest and each band will be one base different in size from the one below it. In the example given the sequence of the DNA will read – ACCCTTGG.
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USING THIS APPROACH WHAT HAS BEEN WORKED OUT
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Using this approach the sequence of all human genes has been worked out. We have learned how to read genes and to some extent work out what they do. This is a sequence for the alpha globin gene.
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WHAT CAN WE RECOGNISE
AND WE CAN ALSO RECOGNISE THE STOP SIGNAL |
THE START SIGNAL
THE STOP SIGNAL |
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WHAT IS THE UK GOVT CURRENTLY FUNDING
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The U.K. government is currently funding a major initiative for genetic screening and counselling The Wellcome Trust and The Department of Health – co funding a study on Genetics Education and training across all the non-genetic clinical professions. So it would appear that we are making great strides in the application of this research for the good of man.
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WHAT IS THE ROLE OF THIS TECH IN GENETIC DIAGNOSIS
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But what is the role of this technology in genetic diagnosis? Pre natal screening can be used to detect genetic defects in unborn foetuses. This can result in parents being offered a termination of pregnancy if they feel they cannot cope with a genetically damaged child. For many polygenic disorders screening can be used to produce a risk assessment for an individual in order that he or she may make lifestyle changes to minimise risk. We are briefly going to look at some of the ethical aspects of screening and to consider potential sociopolitical impacts.
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ADVANTAGES OF SCREENING
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There are clearly some advantages to using these approaches to screening e.g Genetic differences in drug metabolism can be identified to maximise therapeutic benefits Adverse drug reactions – at the moment these seem idiosyncratic, but is there a scientific basis that we can determine through genetic screening Similarly Different responses to medicines – can we use genetic predictors to see who is most likely to respond to treatment And can we Identify “at risk groups” and suggest lifestyle change
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POTENTIAL RISKS OF SCREENING
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There are clear disadvantages too: The expense of Mass screening Misuse of data: health insurance, healthcare provision Socially stigmatising Will individuals react inappropriately to test results – depression/suicide for negative findings or even ignoring health warnings, smoking and drinking more if “good genes” identified
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WHAT IS LIKELY WITH REGARDS TO SCREENING
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Commercial control of health screening Available only to the wealthy? Combining genetic makeup with chosen lifestyle to determine future health Little emphasis on major factors such as poverty or material wealth
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LONGER TERM ISSUES WITH SCREENING
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Genetic screening will lead to selection of favoured genes. This will lead to loss of diversity in the population Diversity is a protective mechanism for a species e.g. ability to adapt or survive pandemic contagious diseases N.B remember the advantage of carrying one gene for sickle cell disease in an area where malaria is endemic.
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