4: Manipulation of Nucleic Acids

Restriction enzymes, gel electrophoresis, and PCR.

DNA Fragment Size in Agarose Gel Electrophoresis

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

• A. 150 base pairs
• B. 400 base pairs
• C. 1,000 base pairs
• D. 7,000 base pairs

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Determining DNA Size from Gel Electrophoresis

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Gel Migration Problem

DNA Marker	# of Base Pairs (bp)	Migration Distance (cm)
500 base pairs	500	3.01
1,000 base pairs	1000	2.75
2,000 base pairs	2000	2.49
3,000 base pairs	3000	2.34
5,000 base pairs	5000	2.15
10,000 base pairs	10000	1.88
Unknown	? ?	1.97

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 16% of the correct number to be correct.

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Determining Inverse PCR Primer Selection

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

  U

  U

  A

  ,

  C

  C

  G

  –3′

  AND  

  5′–

  A

  U

  G

  ,

  C

  G

  A

  –3′

• B.  

  5′–

  G

  C

  C

  ,

  A

  U

  U

  –3′

  AND  

  5′–

  A

  U

  G

  ,

  C

  G

  A

  –3′

• C.  

  5′–

  U

  C

  G

  ,

  C

  A

  U

  –3′

  AND  

  5′–

  U

  A

  C

  ,

  G

  C

  U

  –3′

• D.  

  5′–

  U

  U

  A

  ,

  C

  C

  G

  –3′

  AND  

  5′–

  A

  G

  C

  ,

  G

  U

  A

  –3′

• E.  

  5′–

  A

  G

  C

  ,

  G

  U

  A

  –3′

  AND  

  5′–

  G

  C

  C

  ,

  A

  U

  U

  –3′

• F.  

  5′–

  G

  C

  C

  ,

  A

  U

  U

  –3′

  AND  

  5′–

  C

  G

  G

  ,

  U

  A

  A

  –3′

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Determining Restriction Fragment Sizes

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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.

					MssI				BsnI						MssI
	0		1		2		3		4		5		6		7		8		9		10		11		12

Two (2) distinct types of restriction enzyme recognition sites, MssI and BsnI, 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 MssI only.

• A. 1 kb
• B. 2 kb
• C. 3 kb
• D. 4 kb
• E. 5 kb
• F. 6 kb

Check Answer Clear Selection

 

Determining Nested PCR Primer Pairs

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

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

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

The amplicon sequence of DNA shown above was replicated using 30 cycles of PCR, using the primers 5′-GACGAT-3′ and 5′-TTCTAC-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′–

  G

  C

  U

  ,

  C

  U

  A

  –3′

  AND  

  5′–

  A

  C

  G

  ,

  U

  C

  C

  –3′

• B.  

  5′–

  U

  A

  G

  ,

  A

  G

  C

  –3′

  AND  

  5′–

  C

  C

  U

  ,

  G

  C

  A

  –3′

• C.  

  5′–

  C

  C

  U

  ,

  G

  C

  A

  –3′

  AND  

  5′–

  G

  C

  U

  ,

  C

  U

  A

  –3′

• D.  

  5′–

  A

  U

  C

  ,

  U

  C

  G

  –3′

  AND  

  5′–

  C

  C

  U

  ,

  G

  C

  A

  –3′

• E.  

  5′–

  A

  U

  C

  ,

  U

  C

  G

  –3′

  AND  

  5′–

  A

  C

  G

  ,

  U

  C

  C

  –3′

• F.  

  5′–

  U

  G

  C

  ,

  A

  G

  G

  –3′

  AND  

  5′–

  U

  A

  G

  ,

  A

  G

  C

  –3′

Check Answer