DNA Sequencing Flashcards
Define the term DNA sequencing
-a technique that allows genes to be isolated and read
What are the 2 key methods for DNA sequencing
1) Sanger sequencing
2) Pyrosequencing
What are the 5 ingredients needed for Sanger sequencing
1) DNA polymerase enzyme
2) primer
3) four DNA nucleotide
4) template DNA to be sequenced
5) dideoxynucleotide
What is DNA polymerase enzyme used for in Sanger sequencing
-to catalyse the addition of the free nucleotides
What is a primer and what is its role in Sanger sequencing
-a short piece of single-stranded DNA
-its role is to bind to the template DNA and act as a “starter” for the polymerase
What is the role of the dideoxynucleotides
-they are chain terminating versions of the four nucleotides and end the sequencing when present
How come dideoxynucleotides end sequencing
-because they lack an -OH group on carbon 3 so they don’t allow for replication to be continued
Explain the method of Sanger sequencing
1) ingredients are added to a tube
2) the mixture is heated up (to separate the phosphodiester bonds between the strands)
3) it is then cooled so that the primer can bind to the single-stranded template
4) DNA polymerase synthesises new DNA starting from the primer
5) DNA polymerase continues adding nucleotide to the chain until it happens to add a dideoxynucleotide instead of a normal one, so no further nucleotides can be added so the strand ends with the dideoxynucleotide
6) process repeats itself in number of cycles
7) the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA
8) the ends of the fragments will be labelled with radioactive primer
9) these fragments are then sorted into size order by electrophoresis
What does the Sanger sequence ensure
-that a dideoxynucleotide will have been incorporated at every single position of the target DNA in at least one reaction
What does the Sanger sequencing method rely on
-the chain terminating (dideoxynucleotides) being incorporated
What is a disadvantage of Sanger sequencing
-it is very slow
Explain cloning DNA using a vector
1) GENE ISOLATION
->specific gene from a bacterium is cut out using restriction enzymes, which recognise and cut specific DNA sequences
2) INSERTION INTO A VECTOR
->the isolated gene is inserted into a bacterial plasmid (vector)
3) TRANSFORMATION INTO A HOST BACTERIA
-> the genetically modified plasmid is introduced into E. coli
4) BACTERIAL REPLICATION AND GENE AMPLIFICATION
->as the E. coli cells divide they replicate the plasmid, making many copies of the inserted gene
5) PLASMID ISOLATION
->the bacteria are cultured and the plasmids containing the gene of interest are extracted using plasmid preparation techniques
6) DNA SEQUENCING
->the isolated DNA is then sequenced to determine its nucleotide order which allows researchers to analyse the genes structure and function
What does pyrosequencing use on its terminal bases compared to Sanger sequencing
-pyrosequencing uses fluorescent dyes instead of radioactivity
How can scientist identify terminal bases in pyrosequencing
-they scan strands with laser beam and the dyes glow, the light signature was identified by computer
What piece of equipment is used to read pyrosequences
-autoradiograms
How does pyrosequencing use sequencing
-by synthesis
Summarise what pyrosequencing involves
-synthesising a single strand of DNA, complementary to the strand to be sequenced, one base at a time whilst detecting by light emission, which base was added at each step
What are the 9 ingredients needed in pyrosequencing
1) DNA sample
2) primer
3) DNA polymerase
4) modified nucleotides
5) ATP sulfurylase
6) luciferase
7) apyrase
8) luciferin
9) APS (adenosine phosphosulfate)
What is ATP sulfurylase
-an enzyme that forms ATP from adenosine phosphosulfate (APS) and pyrophosphate (PPi)
What is luciferase
-an ATPase that catalyses the conversion
What is apyrase
- removes unincorporated nucleotides (nucleotides that were not added to the growing DNA strand during DNA synthesis)
What is luciferin
-a generic term for the light-emitting compound found in organisms that generate bioluminescence
Explain the method of pyrosequencing
1) long piece of DNA is cut into fragments using a nebuliser
2) these lengths are degraded to single stranded DNA
3) all ingredients are added- but only 1 of the 4 possible activated nucleotides is added at any one time
4) when a modified nucleotide is added, the two extra phosphorylase are released as pyrophosphate (PPi)
5) in the presence of APS, the enzyme ATP sulfurylase converts PPi to ATP
6) in the presences of ATP, the enzyme luciferase converts luciferin to oxyluciferin which generates visible light that is detected by a camera
7) the amount of light generated is proportional to the amount of ATP available and therefore indicated how many of the same type of activated nucleotides were incorporated adjacently
8) this is recorded on a pyrograph
9) unincorporated nucleotides are degraded by apyrase
What size of DNA can be sequenced in Sanger sequencing
Large
What size of DNA can be sequenced in pyrosequencing
-small fragments
Are dideoxynucleotides used in Sanger sequencing
-yes
Are dideoxynucleotides used in pyrosequencing
-No
Are radioactive primers used in Sanger sequencing
-yes
Are radioactive primers used in pyrosequencing
-no
Is DNA polymerase used in Sanger sequencing
-yes
Is DNA polymerase used in pyrosequencing
-yes
Is Sanger sequencing a chain terminating sequence
-yes
Is pyrosequencing a chain terminating process
-no
Is there production of light in Sanger sequencing ing
-no
Is there production of light in pyrosequencing
-yes
Is gel electrophoresis used in Sanger sequencing
-yes
Is gel electrophoresis used in pyrosequencing
-no
What is the relative speed of Sanger sequencing
-slow
What is the relative speed of pyrosequencing
-faster
Is Sanger sequencing cheap or expensive
-expensive
Is pyrosequencing cheap or expensive
-cheap
What does DNA sequencing enables us to do
-enables us to make comparisons between organisms of the same and different species
True or false:
A few genes are unique to humans
-true
-most of our genes have counterparts with other organisms
What does the fact that most of our genes have counter parts with other organisms verify
-verifies that genes that work well tend to be conserved by evolution
-some genes are co-opted to perform new tasks
What are the places on the DNA where substitutions occur called
-single nucleotide polymorphisms (SNPs)
What is synthetic biology
-an interdisciplinary science concerned with designing and building useful biological devices and systems
What are examples of aspects of synthetic biology
-biomedicine
-biofuels
-food production
-chemicals
-biomaterials and biosensors
What is the role of PCR (polymerase chain reaction)
-amplifying & cloning small amount of DNA (found at crime scenes) to make thousands of millions of copies
Who was PCR first discovered by
-Karry Mullis
Why do we use PCR
-so we have enough DNA to do multiple tests
What 4 factors does PCR rely on
->that DNA is made up of 2 anti-parallel back bone strand
->each strand has a 3’ and 5’ end
->DNA is only synthesised from the 3’ end
->bases pair up with complementary bases
What are the 3 main differences between PCR and DNA replication
->only short sequences are replicated and not entire chromosomes
->requires the addition of a primer
->a cycle of heating and cooling is required to separate DNA strands, bind primers and for replication
What are the 5 key reagents in PCR
1) original DNA sample
2) primers
3) free nucleotides
4) DNA Taq
5) PCR machine
What is the use of the original DNA sample in PCR
-as it is what we are trying to amplify
What is the use of primers in PCR
-it directs where the polymerase starts
What is the use of free nucleotides in PCR
-for complementary base pairing
What is the use of DNA Taq polymerase in PCR
-specialised polymerase, needed as it survives all temperatures
What is the use of the PCR machine in PCR
-its a thermal cycler, which regulates different temperatures needed for different stages
Describe what happens in PCR
1) the sample of DNA is mixed with DNA nucleotides, primers, magnesium ions and the enzyme Taq DNA polymerase
2) mixture is heated to around 94-96°C to break the hydrogen bonds between the complementary nucleotide base pairs and so denatures the double stranded DNA into two single strands of DNA
3) mixture is cooled to around 68°C, so the primers can bind to one end of each single strand
4) the Taq DNA polymerase enzyme molecule can now bind to the end where there is double-stranded DNA. Taq polymerase can withstand high temp (72°C)
5) the temp is raised to 72°C which keeps the DNA as single strands
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) whole process begins again and is repeated for many cycles
How does the amount of DNA increase in PCR
1 —> 2 —> 4 —> 8 —> 16 —> 32 —> 64 —> 128 and so on
What are the 7 applications of PCR
1) tissue typing
2) detection of oncogenes
3) detecting mutations
4) identifying viral infections
5) monitoring the spread of infectious disease
6) forensic science
7) research
What is the name of the enzyme that bacteria contain
-restriction endonuclease
What are restriction enzymes used for
-enables us to isolate genes
How do bacteria protect themselves from attack by bacterial viruses
-because they contain enzymes that ‘cut up’ or splice the viral DNA
