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
Is PCR an exponential amplification?
Yes
Is it true that PCR has both specificity and sensitivity?
Yes
How does PCR have specificity?
This is provided by the primers. They are complementary to opposite strands with their 3’ ends pointing towards each other.
How does PCR have sensitivity?
one target molecule can be amplified to > 109 molecules in just a few hours, because a product formed in one cycle becomes a template for the next.
what are the applications of PCR?
- Making specific mutations
- Sequencing of archaic hominin genomes
- Forensic science
Who found out that VNTRs can be used to identify people?
Sir Alec Jeffreys in 1984
Describe a VNTR
- VNTRs are short DNA sequences (10–100 base pairs) that repeat multiple times in a row. The number of repeats varies greatly between individuals, making VNTRs highly unique for each person.
What is an STR?
STRs are repetitive DNA sequences that vary in number between individuals. Each individual inherits STRs from their parents, providing a unique DNA profile.
Is it true that an STR can be used for personal and parental identification ?
Yes
Why isn’t just a single STR useful?
Because a single STR may be shared by unrelated people.
What is CODIS ?
- A multiplex PCR is performed (multiple reactions in one tube) using specific primers for 13 different STRs on different chromosomes. Allows for human identification.
- European standards overlap with CODIS for global compatibility.
After 30 cycles of PCR, how many copies of target DNA can be obtained?
Over 1 billion copies of the DNA target can be obtained.
Insights from Neanderthals and Denisovans
- DNA sequencing of Neanderthal fossils showed modern non-African humans share ~2% of their DNA.
- Denisovan DNA was identified in a cave, revealing interbreeding with ancestors of Melanesians and some Asians.
- Interbreeding events were localized in areas like the Arabian Plate.
Fossil and genetic evidence suggest the existence of which several human-like species?
- Homo erectus: Early ancestor, possibly contributing DNA to later populations.
- Homo floresiensis: The “hobbit” species, limited to Indonesian islands.
- Denisovans: Known from sparse remains, genetically distinct but related to Neanderthals.
Describe how to do forensic DNA profiling?
- Design primers to flank STR regions.
- Use PCR to amplify STRs.
- Compare sizes of amplified DNA fragments using electrophoresis.
is STR-based DNA profiling used for identifying individuals and for solving crimes?
Yes
