Lecture 10: DNA Sequencing and PCR [G]

Question 1

What does DNA sequencing do?

Answer 1

It determines the order of nucleotides/ bases in DNA.

Question 2

Who got a joint noble prize for their work in DNA sequencing?

Answer 2

Walter GIlbert and Frederick Sanger

Question 3

What is a dideoxynucleotide (ddNTP) ?

Answer 3

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.)

Question 4

What does Sanger DNA sequencing rely on?

Answer 4

the incorporation of dideoxynucleotides into newly replicated DNA

Question 5

Name a dideoxynucleotide (ddNTP)

Answer 5

2’,3’-Dideoxyribonucleoside triphosphate, NTP

Question 6

In a dideoxynucleotide what is the hydroxyl group (-OH) on the 3’ carbon of the sugar replaced by?

Answer 6

A hydrogen atom

Question 7

Why are dideoxynucleotides terminators?

Answer 7

ddNTPs have no 3’-OH group. When incorporated into DNA, no more nucleotides can be added. Further elongation is prevented.

Question 8

Describe the principle of Sanger’s dideoxy sequencing method

Answer 8

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)

Question 9

Deoxynucleotide is the nucleotide usde in DNA.

Answer 9

Contains deoxyribose sugar

Question 10

Describe Sanger’s sequencing method in practice

Answer 10

• 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.

Question 11

Describe Automated dideoxy sequencing

(sanger’s renewed dna sequencing method)

Answer 11

• 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

Question 12

What were the benefits of Automated dideoxy sequencing?

Answer 12

• 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.

Question 13

Describe the process of PCR

Answer 13

• 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.

Question 14

What does PCR do?

Answer 14

It amplifies a specific DNA sequence

Question 15

Who invented PCR?

Answer 15

Kary Mullis in 1983, resulting in his Nobel Prize in Chemistry.

Question 16

When did Watson and Crick come up with their double-stranded DNA model?

Answer 16

In 1953

Question 17

When did Arthur Kornberg discover the first DNA polymerase (DNA Polymerase I in E.coli) ?

Answer 17

In 1957

Question 18

When did Har Gobind Khorana started deciphering the genetic code?

Answer 18

In the 1960s

Question 19

When did homas Brock isolated a bacterium, Thermus aquaticus, rom a hot spring in Yellowstone National Park. ?

Answer 19

In 1969

Question 20

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). ?

Answer 20

In 1971

Question 21

When was Taq polymerase was isolated from T. aquaticus.?

Answer 21

in 1976

Question 22

When did Sanger get his nobel prize for ddDNA sequencing?

Answer 22

In 1977

Question 23

What is a useful feature of Taq polymerase?

Answer 23

It is thermally stable and thrives at ~ 72 degrees celcius.

Question 24

Describe the process of agarose gel electorphoresis

Answer 24

• 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

Question 25

Is PCR an exponential amplification?

Answer 25

Yes

Question 27

Is it true that PCR has both specificity and sensitivity?

Answer 27

Yes

Question 28

How does PCR have specificity?

Answer 28

This is provided by the primers. They are complementary to opposite strands with their 3’ ends pointing towards each other.

Question 29

How does PCR have sensitivity?

Answer 29

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.

Question 30

what are the applications of PCR?

Answer 30

• Making specific mutations
• Sequencing of archaic hominin genomes
• Forensic science

Question 31

Who found out that VNTRs can be used to identify people?

Answer 31

Sir Alec Jeffreys in 1984

Question 32

Describe a VNTR

Answer 32

• 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.

Question 33

What is an STR?

Answer 33

STRs are repetitive DNA sequences that vary in number between individuals. Each individual inherits STRs from their parents, providing a unique DNA profile.

Question 34

Is it true that an STR can be used for personal and parental identification ?

Answer 34

Yes

Question 35

Why isn’t just a single STR useful?

Answer 35

Because a single STR may be shared by unrelated people.

Question 36

What is CODIS ?

Answer 36

• 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.

Question 37

After 30 cycles of PCR, how many copies of target DNA can be obtained?

Answer 37

Over 1 billion copies of the DNA target can be obtained.

Question 38

Insights from Neanderthals and Denisovans

Answer 38

• 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.

Question 39

Fossil and genetic evidence suggest the existence of which several human-like species?

Answer 39

• 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.

Question 40

Describe how to do forensic DNA profiling?

Answer 40

• Design primers to flank STR regions.
• Use PCR to amplify STRs.
• Compare sizes of amplified DNA fragments using electrophoresis.

Question 41

is STR-based DNA profiling used for identifying individuals and for solving crimes?

Answer 41

Yes