Bio Techniques

Question 1

How DNA sequence data is obtained for genomic research?

Answer 1

1. Obtain Samples
2. Purify the DNA
3. Copy your Gene
4. Make sure your gene is copied
5. Obtain sequence data

Question 2

DNA Purification Using “Spin
Columns”

Answer 2

1. Chop up tissues and break open the cells with detergents
2. Separate the DNA from the rest of the cell debris using spin column and centrifugation
3. Suspend the DNA in buffer for future use

Question 3

Common challenges in DNA extraction

Answer 3

1. Sample Type
2. Sample Preparation and Storage
3. Choosing the Right Method

Question 4

Polymerase Chain Reaction (PCR)

Answer 4

technique allowing the exponential amplification of small quantities/specific regions of DNA utilizing thermal cycling and enzymatic reactions

Question 5

PCR is developed by

Answer 5

Kary Mullis in 1983

Question 6

PCR components

Answer 6

• Template (DNA)
• Primers
• DNA polymerase (Taq)
• dNTPs (deoxynucleotide triphosphate)
• Buffers

Question 7

PCR Process

Answer 7

• Initialization
• Denaturation
• Annealing
• Extension
• Repeat
• Final Elongation
• Hold

Question 8

Choosing primers:

Answer 8

• Should be 18-25 nucleotides in length
• Calculated melting temperature varies depending on the method used (55-65°C), but should be nearly identical for both primers
• Avoid inverted repeat sequences and self-complementary sequences in the primers, avoid complementarity between primers (‘primer dimers’)
• Have a G or C at the 3’ end (a G/C “clamp”)

Question 9

Sources of problem in PCR:

Answer 9

• Inhibitors of the reaction from the template DNA preparation (protease, phenol, EDTA, etc)
• Cross-contamination by DNA from sources other than the template added

Question 10

if this becomes a problem:

Answer 10

• Work in a laminar flow hood (decontaminate using UV light 254 nm)
• Use PCR dedicated pipettors (with barrier tips), PCR dedicated reagents
• Centrifuge tubes before opening them to prevent spattering, pipet contamination

Question 11

Types of PCR:

Answer 11

• End-point PCR
• Reverse-Transcriptase PCR (RT-PDR)
• Multiplex PCR
• Quantitative of real-time PCR (qPCR)

Question 12

End-point PCR, end products?

Answer 12

are visualized on an agarose gel to determine their size as well as relative quantity

Question 13

End-point PCR, is used for?

Answer 13

applications such as cloning, sequencing, genotyping and sequence detection

Question 14

Endpoint PCR is ____ ___ ____ than real-time PCR

Answer 14

far less quantitative

Question 15

End-point PCR is mostly used to

Answer 15

• detect presence or absence of targets
• but can also be used to estimate relative quantity

Question 16

Reverse-Transcriptase PCR (RT-PCR)

Answer 16

technology by which RNA molecules are converted into their complementary DNA (cDNA) sequences by reverse transcriptases

Question 17

Multiplex PCR

Answer 17

• used for the amplification of multiple targets in a single PCR experiment
• it amplifies many different DNA sequences simultaneously

Question 18

Quantitative or real-time PCR (qPCR)

Answer 18

• the DNA amplification is detected in real-time with the help of a fluorescent reporter
• the signal strength of the fluorescent reporter is directly proportional to the number of amplified DNA molecules

Question 19

Agarose gel electrophoresis

Answer 19

1. Determine the required gel percentage
2. Pour the gel
3. Mix samples/ladders with loading dye
4. Load the gel
5. Run the gel
6. Visualization

Question 20

Can you identify the purpose of the polymerase chain reaction?

Answer 20

It is a DNA amplification technique

Question 21

DNA extraction from plant tissues is difficult due to:

Answer 21

Presence of secondary metabolites and polysaccharides

Question 22

DNA extraction requires _________to lyse the epithelial cells
and to degrade compounds inhibitory to amplification

Answer 22

Heat treatment

Question 23

In the agarose gel the DNA migrates in the following direction:

Answer 23

negative to positive pole

Question 24

The ratio of absorption at 260nm to absorption at 280nm is commonly used to assess:

Answer 24

The purity of DNA and RNA with respect to protein

Question 25

What are the fundamental requirements of a PCR reaction?

Answer 25

• DNA segment to be amplified
• Two oligonucleotide primers
• A heat-stable DNA polymerase

Question 26

Which of the following A260/280 ratios would be a reasonable cut off for RNA that you intend to use for quantitative RT-PCR?

Answer 26

> 1.8

Question 27

Which of the following is not a method to remove contaminant protein from sample solution?

Answer 27

Washing with buffer containing EDTA

Question 28

Which of the following reagents are used for precipitating DNA?

Answer 28

• ethanol
• isopropanol

Question 29

Which of these dyes could you use to visualise DNA run on an agarose gel?

Answer 29

Ethidium Bromide