Lecture 10: DNA Sequencing and PCR [G] Flashcards
Wednesday 16th October
What does DNA sequencing do?
It determines the order of nucleotides/ bases in DNA.
Who got a joint noble prize for their work in DNA sequencing?
Walter GIlbert and Frederick Sanger
What is a dideoxynucleotide (ddNTP) ?
A modified nucleotide that prevents DNA from elongating further by terminating the chain at a specific nucleotide (ddNTPs lack a 3’-hydroxyl group, preventing further extension of DNA.)
What does Sanger DNA sequencing rely on?
the incorporation of dideoxynucleotides into newly replicated DNA
Name a dideoxynucleotide (ddNTP)
2’,3’-Dideoxyribonucleoside triphosphate, NTP
In a dideoxynucleotide what is the hydroxyl group (-OH) on the 3’ carbon of the sugar replaced by?
A hydrogen atom
Why are dideoxynucleotides terminators?
ddNTPs have no 3’-OH group. When incorporated into DNA, no more nucleotides can be added. Further elongation is prevented.
Describe the principle of Sanger’s dideoxy sequencing method
Add:
- A single stranded template DNA
- A primer complementary to part of this template
- DNA polymerase
- A pool of normal deoxynucleotides (dATP, dTTP, DGTP and dCTP)
- A small proportion of radioactively-labelled ddATP
- Some newly synthesized molecules will get a ddATP in each place that there is a T in the template DNA.
- The result is a nested set of new DNA molecules each of which ends in a ddA. (base is adenine)
Deoxynucleotide is the nucleotide usde in DNA.
Contains deoxyribose sugar
Describe Sanger’s sequencing method in practice
- Take the template (lower strand), primer, DNA polymerase, and the dNTP’s and divide into 4 aloquots.
- Add the appropriate ddNTP to each test tube. ddG to one, ddA to one, ddT to one, ddC to one.
- If you put the nested series next to each other you can separate them on the basis of size using electropheresis.
- Autoradiograph and read sequence from the bottom upwards.
Describe Automated dideoxy sequencing
(sanger’s renewed dna sequencing method)
- ① template + primer + DNA Pol + all dNTPs + all fluorescent ddNTPs in ONE tube
- ②The dye present in each synthesized fragment corresponds to the dye attached to the dideoxynucleotide that terminated the synthesis of that particular fragment.
- ③ Pass nested products through an electrophoretic system and read with lasers
What were the benefits of Automated dideoxy sequencing?
- Automation with fluorescent dyes tagged on ddNTPs:
- Allowed sequencing to be done in a single reaction tube.
- Capillary electrophoresis and lasers replaced manual gel reading.
- Dramatically increased speed, accuracy, and affordability.
Describe the process of PCR
- Denaturation (94°C): Double-stranded DNA is heated to separate into single strands.
- Annealing (50-60°C): Short primers bind specifically to complementary sequences on the DNA.
- Extension (72°C): A thermostable enzyme (e.g., Taq polymerase) synthesizes new DNA strands starting at the primers.
- Each cycle doubles the amount of target DNA.
- Amplified DNA is loaded onto an agarose gel for electrophoresis.
- DNA migrates based on size, with smaller fragments moving faster.
- Visualization: Gels are stained with fluorescent dyes and imaged under UV light.
What does PCR do?
It amplifies a specific DNA sequence
Who invented PCR?
Kary Mullis in 1983, resulting in his Nobel Prize in Chemistry.
When did Watson and Crick come up with their double-stranded DNA model?
In 1953
When did Arthur Kornberg discover the first DNA polymerase (DNA Polymerase I in E.coli) ?
In 1957
When did Har Gobind Khorana started deciphering the genetic code?
In the 1960s
When did homas Brock isolated a bacterium, Thermus aquaticus, rom a hot spring in Yellowstone National Park. ?
In 1969
WHen did Khorana’s group suggested that a template-primer-polymerase system could be used for copying DNA … and using two primers should lead to replication of a specific fragment of DNA (but did not show results). ?
In 1971
When was Taq polymerase was isolated from T. aquaticus.?
in 1976
When did Sanger get his nobel prize for ddDNA sequencing?
In 1977
What is a useful feature of Taq polymerase?
It is thermally stable and thrives at ~ 72 degrees celcius.
Describe the process of agarose gel electorphoresis
- The reactions are loaded into the wells of a slab gel made of agarose.
- An electric current is applied.
- DNA is negatively charged, so it migrates to the positive terminal.
- The gel contains a dye that binds DNA, so the DNA will fluoresce under ultraviolet light
- Large fragments migrate SLOWLY: small fragments QUICKLY
