Using Gene Sequencing Flashcards
What is the genome?
The total of all the genetic material in an organism
Where is DNA found in prokaryotes?
In the cytoplasm. Both in the main chromosome and the plasmids
Where is DNA found in Eukaryotes?
In the nucleus of the cell and in mitochondria and in green plant cells DNA is also in the chloroplasts
What are exons?
The coding regions of DNA (the genes)
What are introns?
The large non-coding regions of DNA that are removed before messenger RNA is translated into proteins
What is DNA/ gene sequencing?
The analysis of the individual base sequence along a DNA strand or an individual gene
What do you do in gene sequencing?
Analyse individual strands of DNA or individual genes, giving us a pattern of bases that codes for a particular protein in the cell
What do you do in DNA profiling?
Analyse the patterns in the non-coding areas of DNA (introns) and use them to identify individuals
What is the Polymerase chain reaction (PCR) used to do?
Amplify a sample of DNA to make more genetically identical copies of it very rapidly
Wwhat does it mean when we say that DNA has been amplified?
It is replicated repeatedly using the polymerase chain reaction to produce a much bigger sample
When would you need to use PCR to amplify DNA?
In a crime investigation there may only be a minute DNA sample available and you need at least 1ug of DNA for DNA profiling (equivalent of DNA from 10 000 human cells)
Why did scientists initially find it hard to amplify tiny amounts of genetic material?
The DNA sample needed to be heated to around 90-95°c to seperate the strands. However this destroys the DNA polymerase from most organisms
Kary Mullis solved the problem of developing PCR. How did he do this?
He used enzymes from a bacterium that lives in hot springs. Because the enzymes in this bacterium has evolved to survive in the extreme conditions he hypothesised that they would be robust enough to cope with the high temperatures needed to seperate the DNA strands
What are the stages of PCR?
1) the following are added to the thermocycler:
- the DNA fragment to be copied
- Taq DNA polymerase
- nucleotides
- primers
2) the thermocycler heats the mixture up to 95°c. This causes the hydrogen bonds between the bases to break causing the 2 strands to seperate
3) the mixture is cooled to 55°c. This causes the primers to join with their complementary base pairs at the end of each DNA strand. DNA polymerase attaches to the double strands.
4) - the temperature is increased to 72°c this is the optimum temperature for DNA polymerase. It adds complementary each DNA strand. Once the 2 strands are completed the process is repeated. The number of DNA strands increase exponentionally. Each cycle takes around 2 mins to complete
What are primers?
Small sequences of DNA that must join to the beginning of seperated DNA strands before copying can begin
What are the basic principles seen in all the different methods of DNA sequencing?
- the DNA strands are chopped into smaller pieces
- the double strands are seperated to give single strands
- PCR is involved in replicating the DNA fragments to produce large quantities of material for analysis
- labelled terminator bases are added to the single strands of DNA. A terminator base is a modified version of one of the A,T,C,G nucleotide bases. When a terminator base is incorporated into a DNA molecule the chain is halted as no more bases can be added. A T terminator stops DNA synthesis where a T base would be added ect. Each type of terminator base has a fluorescent tag in a specific colour
- the coloured tags enable the sequence of bases to be read very rapidly by an automated system
What is massively parallel sequencing?
A very rapid method of sequencing millions of DNA fragments at the same time
What are current sequencing technologies heavily dependent on?
State of the art computer technology. The raw data is fed into computer systems that can reassemble the genomes. They compare the fragments and find the areas of overlap between them
How can scientists use DNA sequencing to determine the protein produced from any gene and what realisation has this led to?
Because of the universal code we can recognise the start and stop codons in a gene. Analysing the base pairs enables us to work out which amino acids will be joined together to form a protein as a result of information contained in the DNA.
This has led to the realisation that genes will code for more than one protein. And some genes will code for factors that affect the expression of other genes changing the protein product
How can DNA sequencing help us to gain a better understanding of diseases that result from a mutation in a single gene?
DNA sequencing makes it possible to identify a faulty gene, see which bases have changed and understand hoe the changes in the DNA affect the protein produced. This then allows us to begin to work out how changes in the proteins result in the symptoms of the condition
Most diseases (especially non-communicable ones) are the result of the inheritance of gene variants thaf increase the rism if an individual developing a specific disease. What are gene variants?
Different versions of genes, an alternative term for allele
In many cases what is it the interaction between that determines the incidence of disease?
The genome and the environment
What is the 100 000 genome project?
A UK initiative sequencing the genomes of 100 000 people with cancer and rare genetic diseases. It will provide huge amounts of data showing how particular genotypes combined with environmental conditions raise the risk of or cause specific diseases. The genetic data from patients and their families will be matched with clinical data held on DNA databases. This data will be used by scientisrs to identify mutations associated with different diseases and develop new, targeted therapies
What is DNA profiling?
The identification of repeating patterns in the non-coding regions of DNA
