4: Manipulation of Nucleic Acids

Restriction enzymes, gel electrophoresis, and PCR.

Amplicon Copy Number After PCR Rounds

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Starting with genomic DNA, how many copies of the double-stranded amplicon of the exact length are there after 13 rounds of PCR?

• A. 2⁹ = 512 complete copies
• B. 2¹⁰ = 1,024 complete copies
• C. 2¹¹ = 2,048 complete copies
• D. 2¹² = 4,096 complete copies
• E. 2¹³ = 8,192 complete copies

Check Answer

 

DNA Fragment Migration in Agarose Gel Electrophoresis (Closest/Farthest)

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Which one of the following fragments of DNA would migrate closest to the wells during agarose gel electrophoresis?

• A. 80 base pairs
• B. 350 base pairs
• C. 1,000 base pairs
• D. 6,000 base pairs

Check Answer

 

DNA Fragment Size from Agarose Gel Migration (Numeric)

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Gel Migration Problem

DNA Marker	# of Base Pairs (bp)	Migration Distance (cm)
500 base pairs	500	2.80
1,000 base pairs	1000	2.38
2,000 base pairs	2000	1.95
3,000 base pairs	3000	1.71
5,000 base pairs	5000	1.39
10,000 base pairs	10000	0.97
Unknown	? ?	1.82

The standard DNA ladder and unknown DNA strand listed in the table were separated using an agarose gel
Estimate the number of base pairs of the unknown DNA strand.
Note: answers need to be within 10% of the correct number to be correct.

Check Answer

 

Inverse PCR Primer Selection (15 nt)

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EcoR1 overhang	left unknown sequence	central known sequence	right unknown sequence	EcoR1 overhang
5′–      A   A   ,   T   T   C  – 3′–              ,           G  –	–  N   N   N   ,   N   N   N   ,   N   N   N  – –  N   N   N   ,   N   N   N   ,   N   N   N  –	–  C   C   A   ,   T   A   G   ,   T   G   A   ,   C   T   G   ,   A   C   T  – –  G   G   T   ,   A   T   C   ,   A   C   T   ,   G   A   C   ,   T   G   A  –	–  N   N   N   ,   N   N   N   ,   N   N   N  – –  N   N   N   ,   N   N   N   ,   N   N   N  –	–  G           ,              –3′ –  C   T   T   ,   A   A      –5′

Choose the correct pair of RNA primers that will amplify the both the known and unknown region of DNA shown above using inverse PCR.
The RNA primers are 6 bases in length.
Pay close attention to the 5′ and 3′ ends of the primers.

• A.  

  5′–

  C

  U

  A

  ,

  U

  G

  G

  –3′

  AND  

  5′–

  U

  C

  A

  ,

  G

  U

  C

  –3′

• B.  

  5′–

  C

  U

  G

  ,

  A

  C

  U

  –3′

  AND  

  5′–

  G

  G

  U

  ,

  A

  U

  C

  –3′

• C.  

  5′–

  C

  U

  A

  ,

  U

  G

  G

  –3′

  AND  

  5′–

  C

  U

  G

  ,

  A

  C

  U

  –3′

• D.  

  5′–

  U

  C

  A

  ,

  G

  U

  C

  –3′

  AND  

  5′–

  G

  A

  U

  ,

  A

  C

  C

  –3′

• E.  

  5′–

  C

  U

  G

  ,

  A

  C

  U

  –3′

  AND  

  5′–

  G

  A

  C

  ,

  U

  G

  A

  –3′

• F.  

  5′–

  G

  G

  U

  ,

  A

  U

  C

  –3′

  AND  

  5′–

  U

  C

  A

  ,

  G

  U

  C

  –3′

Check Answer

 

Restriction Fragment Sizes (12 Length, 2 Sites)

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DNA Fragment Question: Shown below is a short DNA fragment that is only 12 kb in length. This fragment has been isolated for restriction enzyme analysis.

							FspI										BcuI				FspI
	0		1		2		3		4		5		6		7		8		9		10		11		12

Two (2) distinct types of restriction enzyme recognition sites, FspI and BcuI, are labeled at the top of this DNA segment.

Determine the sizes of the DNA bands that would appear on an agarose gel after digestion with FspI only.

• A. 1 kb
• B. 2 kb
• C. 3 kb
• D. 4 kb
• E. 5 kb
• F. 6 kb
• G. 7 kb
• H. 8 kb

Check Answer Clear Selection

 

Nested PCR Primer Pair Selection (24 nt)

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5′–  T   C   A   ,   T   C   G  – 3′–  A   G   T   ,   A   G   C  –

–  T   A   A   ,   C   G   T   ,   A   A   G   ,   A   A   T   ,   G   C   G   ,   T   A   C   ,   G   G   A   ,   C   T   C  – –  A   T   T   ,   G   C   A   ,   T   T   C   ,   T   T   A   ,   C   G   C   ,   A   T   G   ,   C   C   T   ,   G   A   G  –

–  A   T   G   ,   A   C   C  –3′ –  T   A   C   ,   T   G   G  –5′

The amplicon sequence of DNA shown above was replicated using 30 cycles of PCR, using the primers 5′-TCATCG-3′ and 5′-GGTCAT-3′.
But the first PCR run contained significant contamination due to mispriming. Probably from using too short of primers that were only 6 nucleotide in length.
Choose the correct pair of RNA primers that will amplify the remaining region of DNA inside the old primers using nested PCR. The nested RNA primers are 6 bases in length.
Pay close attention to the 5′ and 3′ ends of the primers.

• A.  

  5′–

  U

  A

  A

  ,

  C

  G

  U

  –3′

  AND  

  5′–

  G

  A

  G

  ,

  U

  C

  C

  –3′

• B.  

  5′–

  U

  G

  C

  ,

  A

  A

  U

  –3′

  AND  

  5′–

  C

  C

  U

  ,

  G

  A

  G

  –3′

• C.  

  5′–

  C

  U

  C

  ,

  A

  G

  G

  –3′

  AND  

  5′–

  A

  U

  U

  ,

  G

  C

  A

  –3′

• D.  

  5′–

  U

  A

  A

  ,

  C

  G

  U

  –3′

  AND  

  5′–

  C

  C

  U

  ,

  G

  A

  G

  –3′

• E.  

  5′–

  U

  G

  C

  ,

  A

  A

  U

  –3′

  AND  

  5′–

  U

  A

  A

  ,

  C

  G

  U

  –3′

• F.  

  5′–

  G

  A

  G

  ,

  U

  C

  C

  –3′

  AND  

  5′–

  U

  G

  C

  ,

  A

  A

  U

  –3′

Check Answer

 

Restriction Enzyme Overhang Sequences

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Restriction enzymes are proteins that cut DNA at specific sequences to produce fragments for further study. These enzymes are obtained from various types of bacteria and have the ability to recognize short nucleotide sequences within a larger DNA molecule.
The restriction enzyme we are focusing on is Alw44I, which is derived from the bacterium Acinetobacter lwoffi RFL44.
Alw44I cuts the DNA sequence as follows: 5'-G|TGCAC-3', where the '|' indicates the cleavage site.
Based on this information, which one of the following sequences below corresponds to the overhang region of the DNA after cleavage by the restriction enzyme Alw44I?

• A. 5'-ACGT-3'
• B. 5'-CACGT-3'
• C. 5'-GTGCA-3'
• D. 5'-TGCA-3'
• E. 5'-TGCAC-3'

Check Answer

 

Restriction Enzyme Cut Types (5', 3', or Blunt)

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Restriction enzymes are proteins that cut DNA at specific sequences to produce fragments for further study. These enzymes are obtained from various types of bacteria and have the ability to recognize short nucleotide sequences within a larger DNA molecule.
The restriction enzyme we are focusing on is SseBI and is obtained from the bacteria Streptomyces species B.
SseBI cuts the DNA sequence as follows: 5'-AGG|CCT-3' where the '|' indicates the cut location.
Based on this info, can you identify the type of cut this enzyme makes?

• A. 3′ overhang
• B. 5′ overhang
• C. blunt

Check Answer

 

Palindromic DNA Sequence Completion

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The following numbered sequences only contains half of a palindromic sequence.
Match the correct lettered sequence that would finish and replace the 'N's in the sequence to make them palindromes.
Letters will be used exactly once.

	Your Choice	Prompt
	Drop Your Choice Here	1.   5′–  G   N   N   N  –3′
	Drop Your Choice Here	2.   5′–  C   G   G   ,   N   N   N  –3′
	Drop Your Choice Here	3.   5′–  C   C   C   G   ,   N   N   N   G  –3′
	Drop Your Choice Here	4.   5′–  N   N   N   C  –3′

Drag one of the choices below:

• A.  

  G

  C

  C

• B.  

  G

  G

  C

• C.  

  C

  G

  G

• D.  

  C

  C

  G

Check Answer Reset Game

 

PCR Primer Selection (36 bp Template, 9-nt Primers)

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5′–

A

A

G

,

A

A

C

,

C

A

T

,

T

C

G

,

T

T

T

,

G

G

G

,

A

G

G

,

A

G

G

,

T

T

C

,

T

T

G

,

A

C

G

,

T

T

G

–3′

3′–

T

T

C

,

T

T

G

,

G

T

A

,

A

G

C

,

A

A

A

,

C

C

C

,

T

C

C

,

T

C

C

,

A

A

G

,

A

A

C

,

T

G

C

,

A

A

C

–5′

Choose the correct pair of RNA primers that will amplify the entire region of DNA shown above using PCR. The RNA primers are 9 bases in length.
Pay close attention to the 5′ and 3′ ends of the primers.

• A.  

  5′–

  A

  A

  G

  ,

  A

  A

  C

  ,

  C

  A

  U

  –3′

  AND  

  5′–

  A

  A

  C

  ,

  U

  G

  C

  ,

  A

  A

  C

  –3′

• B.  

  5′–

  C

  A

  A

  ,

  C

  G

  U

  ,

  C

  A

  A

  –3′

  AND  

  5′–

  U

  A

  C

  ,

  C

  A

  A

  ,

  G

  A

  A

  –3′

• C.  

  5′–

  A

  A

  G

  ,

  A

  A

  C

  ,

  C

  A

  U

  –3′

  AND  

  5′–

  C

  A

  A

  ,

  C

  G

  U

  ,

  C

  A

  A

  –3′

• D.  

  5′–

  U

  A

  C

  ,

  C

  A

  A

  ,

  G

  A

  A

  –3′

  AND  

  5′–

  A

  U

  G

  ,

  G

  U

  U

  ,

  C

  U

  U

  –3′

• E.  

  5′–

  A

  A

  C

  ,

  U

  G

  C

  ,

  A

  A

  C

  –3′

  AND  

  5′–

  U

  A

  C

  ,

  C

  A

  A

  ,

  G

  A

  A

  –3′

• F.  

  5′–

  U

  A

  C

  ,

  C

  A

  A

  ,

  G

  A

  A

  –3′

  AND  

  5′–

  A

  A

  C

  ,

  U

  G

  C

  ,

  A

  A

  C

  –3′

Check Answer