Manipulating genomes Flashcards
What is DNA sequencing
A technique that allows genes to be isolated and read
How is DNA cloned
1) Gene to be sequenced from a human is isolated using restriction enzymes from a bacterium
2) The DNA is then inserted into the bacterial plasmid (vector)
3) The plasmid is then inserted into the E-Coli host = when cultured it divides many times and the plasmid with the DNA insert will also be copied many times
4) Each new bacterium contains a copy of the gene
5) DNA is then isolated from the bacterium using restriction enzymes
6) The DNA is then made single stranded when the DNA is heated
Fred Sanger - DNA Sequencing is also called
End Termination
Fred Sanger - DNA Sequencing (experiment set up)
1) Used the single strand of DNA as a template for four experiments in separate dishes
2) Each dish contains a solution with the four bases + DNA polymerase
Fred Sanger - DNA Sequencing (experiment process)
3) To each dish a modified version of one of the DNA bases was added, which when incorporated into the synthesised complimentary strand of the DNA no more bases could be further added
- Each modified base was modified with a radioactive isotope
4) As the reaction progressed thousands of the DNA fragments of varying lengths were generated
5) DNA fragments were passed through a gel by electrophoresis
6) Smaller fragments travelled further so fragments could be sorted by length
Fred Sanger - DNA Sequencing
Because fragments were sorted into length order what did this convey
The nucleotide base at the end of each fragment can be read according to its radioactive label, which produces the complimentary base sequence to the DNA
The first sequencing machine
Was based on the Sanger method
-Used fluorescent dyes instead of radioactive isotopes
-Dyes glowed when scanned by a laser beam and the light signature was identified by the computer
-This method dispensed with the need for technicians to read autoradiograms
Autoradiogram
Photographs made when photographic films is exposed to molecules labelled with radioactivity
Primer
Used before DNA polymerase as DNA polymerase needs some DNA to attach too
Pyrosequencing - how is different from Sanger’s method
Uses sequencing by synthesis not by chain termination
= Synthesis of a single strand of DNA complimentary tot he strand being sequenced, whilst detecting by light emission, which base was added at each step
Pyrosequencing
(until primer is added)
1) A long length of DNA is cut into fragments of 300-800 base pairs using a nebuliser
2) These lengths are then degraded into single stranded DNA. These are the template DNAs and they are immobilised
3) Sequencing primer added
Pyrosequencing
(What is the DNA incubated with - step 4)
4) DNA incubated with the enzymes:
- DNA polymerase
-ATP sulfurylase
- Luciferase
-Apyrase
Substrates
-APS
-Luciferin
- One activated nucleotide ( ATP ; TTP ; CTP ; GTP)
What is an activated nucleotide
A nucleotide with two phosphate groups added to it
Pyrosequencing
(What happens after the DNA has been incubated)
5) One of the activated nucleotides (e.g. TTP) is incorporated into the complimentary strand of DNA using the strand to be sequenced as a template
6) The two phosphates are released as pyrophosphate
7) In the presence of APS, ATP sulfurylase converts the pyrophosphate to ATP
8) In the presence of ATP, luciferase converts luciferin to oxyluciferin
9) This conversion generates visible light, which is detected by a camera
Pyrosequencing
What does the amount of light generated indicate
The amount of light is proportional to the amount of ATP available and so indicated how many of the same type of the activated nucleotide was incorporated adjacently in the complimentary DNA strand
- More of the same base = more light
What happens to the unincorporated activated nucleotides
They are degraded by apyrase and the reaction starts again with another nucleotide
Electrophoresis
Process used to separate proteins/DNA fragments of different sizes
-Can separate fragments that differ from only one base pair
Outline electrophoresis set up
-Uses an agarose gel plate covered by a buffer solution
-Electrodes are placed in each of the tank so that when it is connected to the power supply an electric current can pass through
-DNA fragmented by restriction enzymes
-DNA has a negative charge due to many phosphate groups when placed in buffer solution at the cathode the fragments migrate towards the anode (positive electrode)
-Small fragments travel further (need to use a fixed time)
Dyes used
A loading dye to stain it - use a pipette to place in the buffer solution
-After buffer solution is poured away and a dye is added to the gel which adheres to the DNA and stains the fragments
Separating proteins
Similar to DNA but carried out in the presence of a charged detergent such as sodium dodecyl sulphate (SDS) which equalises the surface charge and allows the proteins to separate through the gel according to their molecular mass
- Proteins have a variety of R groups / bonds (all different charges)
What can separating proteins identify
-Sickle cell anaemia: haemoglobin S and not normal haemoglobin A
-Aplastic anaemia, thalassaemia, leukaemia higher then normal amount of fetal haemoglobin and lower amounts of haemoglobin A
What is the polymerase chain reaction (PCR)
A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies
-Used for forensic DNA analysis
What does PCR rely on
-DNA is made of two antiparallel backbone strands
-Each strand has a ‘5 end and a ‘3 end
-DNA only grows on the ‘3 end
-Base pairs pair up according to complementary base pairs A-T C-G
How does PCR differ from DNA replication
-Only short sequences of up to 10,000 base pairs of DNA can be replicated, not entire chromosomes
-Requires the addition of primer for the process to start
-A cycle of heating and cooling is needed to separate the DNA strands; bind primers to the strands and for the DNA to be replicated (cyclic reaction)
Why was initially the process of PCR quite time consuming
Cyclic reaction
-DNA heated to denature then cooled to 35 degrees to anneal the primers and allow the DNA polymerase to work
-Later DNA polymerase was obtained from the thermophilic bacterium - Taq polymerase which is stable at high temperatures (72 degrees is optimum)
Steps of PCR (till cooling)
1) Sample of DNA is mixed with DNA nucleotides; primers; magnesium ions (co-factors) and the enzyme taq polymerase
2) Mixture heated to 96 degrees to break the hydrogen bonds between complementary base pairs and denature the double stranded of DNA into two single strands
3) Mixture is cooled to 68 degrees so primers can anneal to one end of each single strand of DNA
=Small section of double stranded DNA at each end of single stranded DNA molecules
Steps of PCR
4) Taq DNA polymerase can bind to the end where there is double stranded DNA
5) Temperature raised to 72 degrees which keeps DNA single stranded
6) Taq polymerase catalyses the addition of DNA nucleotides starting at the end with the primer and proceeding in a 5’ 3’ direction
7) When Taw DNA polymerase reached the other end of the DNA molecules then a new double strand of DNA has been generated
8) Whole process is repeated
Steps of PCR- how does the DNA increase
Exponentially: 1- 2- 4 -8
After 5 repeats: 2(5)
Anneal
Bind by hydrogen bonds
Application of PCR
-Tissue typing
-Detection of oncogenes
-Detecting mutations
-Identifying viral infections
-Monitoring the spread of infectious disease
-Forensic science
-Research
Tissue typing (PCR)
Donor and recipient tissues can be typed prior to transplantation to reduce the risk of rejection of transplant
Detection of oncogenes (PCR)
If the type of mutation involved in a specific patients cancer is found then the medication may be tailored to that patient
Detecting mutations ( PCR)
A sample of DNA is analysed for the presence of a mutation that leads to a genetic disease
-Parents can be tested to see if they have a recessive allele for a particular gene ; fetal cells may be obtained for the mothers bloodstream for prenatal genetic screening; during IVF treatment one cell from an eight cell embryo can be used to analyse the fetal DNA before implantation
Identifying viral infections (PCR)
Sensitive PCR tests can detect small quantities of viral genome amongst the host cells DNA.
-This can be used to verify HIV
Monitoring the spread of infectious disease (PCR)
The spread of pathogens through a population of wild/ domestic animals / humans can be monitored and the emergence of new more virulent sub-types can be detected
Forensic science (PCR)
Small quantities of DNA can be amplified for DNA profiling to identify criminals or to ascertain parentage
The human genome project
A project in which scientists found that the human genome only contains about 24000 genes
Where are sequenced genomes stored
Gene banks
We share 99% of our genes with the chimpanzee what does this show
Genes that work well will be conserved by evolution
How was pig insulin used to treat patients with diabetes
It is similar to the human genes for insulin
What is FOX -P2
A change in a gene that allowed humans to speak
How are organisms different
Not because their genes are completely different but because some of their shared genes have been altered and now in subtly different ways
-Some changes to the regulatory regions of DNA that do not code directly for proteins have also altered the expression of the genome - the regulatory and coding genes interact in such ways that without he number of genes being increased the number of proteins made may be increased
How can the evolution of certain human pathogens be studied
Bacterial genomes can be extracted from ancient human bones
How are humans different
Same genes but different alleles
Substitution
A mutation which changes DNA sequences between humans
Place where substitutions occur are called SNP’s
-Some have no effect on the protein / alter the protein
-Some can alter the way RNA regulates the expression of another gene
Methylation
Major role in gene regulation
-Can help researchers understand the development of certain human diseases i.e. why does cancer not develop in genetically similar individuals
- Study of this area is called epigenetics
What does the researcher need to know to predict the amino acid sequence
-Have the organisms genome sequenced
-Know which genes code for a specific protein, by using knowledge of which base triplets code for which amino acid
=Determine the primary structure of an amino acid
Also needs to know which parts of the gene codes for introns/exons
Synthetic biology
A science concerned with designing and building useful biological devices and systems
-Encompasses biotechnology, evolutionary biology, molecular biology, systems biology and biophysics
May be used to build engineered biological systems that store ad process information, provide food, maintain human health and enhance the environment
The sequencing of DNA found by analysing genomes provide potential building blocks for synthetic biologists to build devices