W6 - PCR and Sequencing

Question 1

What is the Polymerase Chain Reaction (PCR)?

Answer 1

PCR is a widely used molecular biology technique for amplifying DNA sequences.

Question 2

Why is PCR hailed as one of the monumental scientific techniques of the twentieth century?

Answer 2

PCR is considered monumental because it revolutionized DNA amplification, making it faster and more accessible.

Question 3

How did PCR change the way DNA synthesis was performed?

Answer 3

PCR introduced an in vitro system that moved DNA synthesis from the bacterium to the test tube, making it easier and more controlled.

Question 4

What is the key observation related to the polymerase PolI of the bacteriophage T7 in the context of DNA synthesis?

Answer 4

PolI of the bacteriophage T7 can synthesize double-stranded DNA (dsDNA) from single-stranded DNA (ssDNA) when provided with a short oligonucleotide “primer.”

Question 5

How can you convert dsDNA into ssDNA for further synthesis in the laboratory?

Answer 5

You can achieve this by heating the dsDNA, which will denature it into single-stranded DNA (ssDNA).

Question 6

What role does ssDNA play in the process of in vitro DNA synthesis?

Answer 6

The ssDNA can act as a template for another round of synthesis by enzymes like T7 DNA PolI.

Question 7

What is the temperature limit at which T7 DNA PolI can remain functional?

Answer 7

T7 DNA PolI cannot withstand temperatures as high as 55°C.

Question 8

What temperature is required to completely and terminally inactivate T7 DNA PolI?

Answer 8

85°C

Question 9

Why is the described protocol for in vitro DNA synthesis considered labor-intensive?

Answer 9

It involves multiple steps and temperature manipulations, which require careful attention.

Question 10

Why is it challenging to achieve in vitro DNA synthesis with T7 DNA PolI?

Answer 10

Because using too much T7 DNA PolI can actually inhibit the reaction, making it a delicate balance to optimize the synthesis process.

Question 11

Who is Kary Banks Mullis, and what is he known for in the field of PCR?

Answer 11

Known for his significant contributions to the development of the Polymerase Chain Reaction (PCR) technique.

Question 12

What is the 1st step of PCR?

Answer 12

Denaturation/Melting

At 90°C

Question 13

What is the 2nd step of PCR?

Answer 13

Annealing (adding primers which give specificity to the reactions)

annealing = 2 complementary strands of DNA/RNA come together, form 2 hel

Temperature at which primers bind to DNA = 50-55°C

Question 14

What is the 3rd step of PCR?

Answer 14

Extension - new DNA synthesised by polymerase

Now we have doubled the number of DNA molecules and the cycle repeats, doubling DNA moelcules each time.

Question 15

How can we calculate the number of DNA molecules in a cycle?

Answer 15

no. of DNA molcules = 2^n

n = number of cycles

Question 16

How can we calculate the total number of DNA molecules?

Answer 16

total no. of DNA molcules = no. of starting molecules x 2^n

Question 17

How is PCR done in the lab?

Answer 17

Thermal cyclers (PCR machines)

Question 18

What does a thermal cycler have?

Answer 18

• Heated lid
• Block (48 hole, 96, 2 x 48, 384)
• High ramp rate
• Programmable

Heated lid heats up to 95°C - important as evoporation & condensation can change the overall temp, so high temp prevents accumulation of condensed liquid.

Question 19

What reagents are used in thermal cyclers?

Answer 19

• Taq polymerase
• Taq polymerase buffer, usually with Mg2+
• Forward primer
• Reverse prmer
• dNTPs
• Template
• H2O

Question 20

What is the optimal primer design?

Answer 20

Primers should generally have the following properties:
* Length of 18-24 bases
* 40-60% G/C content
* Start and end with 1-2 G/C pairs
* Melting temperature (Tm) of 50-60°C
* Primer pairs should have a Tm within 5°C of each other
* Primer pairs should not have complementary regions

Question 21

Name a type of primer

Answer 21

Oligonucleotides

Question 22

Why is PCR of a specific region useful?

Answer 22

• Amplifying minute quantities of DNA - e.g. fossil DNA, contaminants, crime scenes, degraded samples…
• Cloning genes
• DNA fingerprinting
• Evaluate gene expression (RT-PCR)
• SNP detection , e.g. Allele-specific PCR or PCR followed by RFLPs
• DNA sequencing

Question 23

Define polymorphic

Answer 23

Regions of human chromosomes that exhibit a great deal of diversity

• variable sequences that can be used to identify individuals

Question 24

Where is the D1S80 locus located?

Answer 24

Located on chromosome 1

• has normally 14-41 repeats of a 16 bp region
• locus = specific location on a chromosome where a particular gene or genetic marker is located
• bp = base pair

Question 25

What is agrose gel electrophoresis?

Answer 25

Method of separating DNA molecules of different sizes.

Question 26

How do you calcuate the length of the tandem repeat?

Answer 26

length of the tandem repeat = n x tandem repeat

Question 27

What are the uses of DNA fingerprinting/profiling?

Answer 27

• Tissue typing: Identifying unknown material (GM food, contaminants, etc), Cancer detection
• Forensic
• Paternity
• Molecular phylogeny

Question 28

What is RT-PCR?

Reverse transcription polymerase chain reaction

Answer 28

Combines reverse transcription and PCR to amplify and analyse RNA molecules.

Question 29

Briefly describe the steps of RT-PCR?

Question 30

What is qPCR?

Quantitative PCR

Answer 30

Quantifies the amount of a specific DNA target sequence in a sample

aka (quantitative) real-time PCR

• allows for the accurate measurement of the initial amount of DNA or RNA in a sample

Question 31

Briefly describe the steps of qPCR?

Question 32

What is ARMS-PCR?

Amplification refractory mutation system /Allele-specific PCR

Answer 32

Allele-specific primers designed to permit the PCR amplification only if the nucleotide at the 3’-end of the primer complemented the base at the wild-type or variant-type DNA sample.

Can be coupled with qPCR

Question 33

Why is DNA sequenced?

Answer 33

• ‘code of life’ – genome projects provide a ‘parts list’ for an entire organism, showing which genes are present.
• Confirm DNA identity
• Identify mutations e.g. causing disease, or insecticide/drug resistance.
• Identify basis of disease

Question 34

What is the Sanger method of DNA sequencing?

aka ‘chain-termination method’ or ‘dideoxy sequencing’

Answer 34

The Sanger method relies on 2’3’dideoxynucleotides (ddNTPs) which lack the 3’ OH required for further nucleotide incorporation by DNA polymerases

Question 35

Next Generation Sequencing/High-throughput sequencing

Answer 35

Faster, massively parallel, less cost, less labour.
Driven by the genome era.

Question 36

What are the differences between Snager and Next Generation Sequencing?

Answer 36

Sanger
* “Each sample must contain a single template eg. cloned piece of DNA. Thus, one sample gives 500 – 1000 bp sequence.”
* Takes days to weeks to sequence a genome depending on size.
* Each read length is usually 500 bp – 1000 bp.

NGS
* “Each sample can be a population and does not require clonal purification. Many different fragments are being sequenced in parallel. Thus, one sample could give up to 3 billion bp (ie. a whole genome).”
* Takes hours to a few days to sequence a genome regardless of size.
* Each read length is about 150 bp.