4: Manipulation of Nucleic Acids

Restriction enzymes, gel electrophoresis, and PCR.

True/False Statements About DNA Structure (BIOL 301)

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Which one of the following statements is TRUE of the structure of DNA?

• A. covalent bonds hold the two strands together
• B. the backbone contains only nucleobase and sugar
• C. the DNA bases for each strand face the outside of the helix
• D. G-C base pairs are stronger than A-T base pairs
• E. the phosphate groups are on the inside of the DNA double helix

Check Answer

 

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 11 rounds of PCR?

• A. 2⁷ = 128 complete copies
• B. 2⁸ = 256 complete copies
• C. 2⁹ = 512 complete copies
• D. 2¹⁰ = 1,024 complete copies
• E. 2¹¹ = 2,048 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 travel furthest from the wells during agarose gel electrophoresis?

• A. 200 base pairs
• B. 400 base pairs
• C. 2,000 base pairs
• D. 5,500 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	2.94
1,000 base pairs	1000	2.50
2,000 base pairs	2000	2.06
3,000 base pairs	3000	1.81
5,000 base pairs	5000	1.49
10,000 base pairs	10000	1.05
Unknown	? ?	2.29

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 18% 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   T   A   ,   A   G   T   ,   T   T   C   ,   C   T   G   ,   G   G   A  – –  G   A   T   ,   T   C   A   ,   A   A   G   ,   G   A   C   ,   C   C   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′–

  C

  U

  G

  ,

  G

  G

  A

  –3′

  AND  

  5′–

  G

  A

  U

  ,

  U

  C

  A

  –3′

• B.  

  5′–

  A

  C

  U

  ,

  U

  A

  G

  –3′

  AND  

  5′–

  A

  G

  G

  ,

  G

  U

  C

  –3′

• C.  

  5′–

  G

  A

  U

  ,

  U

  C

  A

  –3′

  AND  

  5′–

  A

  G

  G

  ,

  G

  U

  C

  –3′

• D.  

  5′–

  G

  A

  C

  ,

  C

  C

  U

  –3′

  AND  

  5′–

  C

  U

  G

  ,

  G

  G

  A

  –3′

• E.  

  5′–

  U

  C

  C

  ,

  C

  A

  G

  –3′

  AND  

  5′–

  G

  A

  C

  ,

  C

  C

  U

  –3′

• F.  

  5′–

  A

  C

  U

  ,

  U

  A

  G

  –3′

  AND  

  5′–

  C

  U

  G

  ,

  G

  G

  A

  –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.

									ChaI		ScaI												ChaI
	0		1		2		3		4		5		6		7		8		9		10		11		12

Two (2) distinct types of restriction enzyme recognition sites, ChaI and ScaI, 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 ChaI 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′–  A   G   G   ,   A   T   C  – 3′–  T   C   C   ,   T   A   G  –

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

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

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

  A

  U

  G

  ,

  C

  A

  G

  –3′

  AND  

  5′–

  G

  U

  A

  ,

  C

  A

  A

  –3′

• B.  

  5′–

  A

  A

  C

  ,

  A

  U

  G

  –3′

  AND  

  5′–

  U

  U

  G

  ,

  U

  A

  C

  –3′

• C.  

  5′–

  G

  U

  A

  ,

  C

  A

  A

  –3′

  AND  

  5′–

  G

  A

  C

  ,

  G

  U

  A

  –3′

• D.  

  5′–

  G

  A

  C

  ,

  G

  U

  A

  –3′

  AND  

  5′–

  A

  A

  C

  ,

  A

  U

  G

  –3′

• E.  

  5′–

  A

  U

  G

  ,

  C

  A

  G

  –3′

  AND  

  5′–

  A

  A

  C

  ,

  A

  U

  G

  –3′

• F.  

  5′–

  A

  U

  G

  ,

  C

  A

  G

  –3′

  AND  

  5′–

  C

  A

  U

  ,

  G

  U

  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 Ksp22I, which is derived from the bacterium Kurthia species 22.
Ksp22I cuts the DNA sequence as follows: 5'-T|GATCA-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 Ksp22I?

• A. 5'-ACTAG-3'
• B. 5'-CTAGT-3'
• C. 5'-GATC-3'
• D. 5'-GATCA-3'
• E. 5'-TGATC-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 MalI and is obtained from the bacteria Marinococus albus I.
MalI cuts the DNA sequence as follows: 5'-GA|TC-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′–  N   N   N   A  –3′
	Drop Your Choice Here	2.   5′–  N   N   N   ,   T   T   A  –3′
	Drop Your Choice Here	3.   5′–  A   N   N   N   ,   A   T   T   T  –3′
	Drop Your Choice Here	4.   5′–  T   A   A   T   ,   N   N   N   A  –3′

Drag one of the choices below:

• A.  

  A

  T

  T

• B.  

  A

  A

  T

• C.  

  T

  A

  A

• D.  

  T

  T

  A

Check Answer Reset Game

 

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

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5′–

T

G

C

,

T

C

T

,

A

C

T

,

T

G

T

,

G

T

G

,

C

C

A

,

A

C

G

,

G

A

G

,

C

A

G

,

C

G

A

,

T

C

T

,

A

T

C

–3′

3′–

A

C

G

,

A

G

A

,

T

G

A

,

A

C

A

,

C

A

C

,

G

G

T

,

T

G

C

,

C

T

C

,

G

T

C

,

G

C

T

,

A

G

A

,

T

A

G

–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′–

  G

  A

  U

  ,

  A

  G

  A

  ,

  U

  C

  G

  –3′

  AND  

  5′–

  U

  C

  A

  ,

  U

  C

  U

  ,

  C

  G

  U

  –3′

• B.  

  5′–

  U

  G

  C

  ,

  U

  C

  U

  ,

  A

  C

  U

  –3′

  AND  

  5′–

  G

  C

  U

  ,

  A

  G

  A

  ,

  U

  A

  G

  –3′

• C.  

  5′–

  G

  A

  U

  ,

  A

  G

  A

  ,

  U

  C

  G

  –3′

  AND  

  5′–

  C

  U

  A

  ,

  U

  C

  U

  ,

  A

  G

  C

  –3′

• D.  

  5′–

  U

  C

  A

  ,

  U

  C

  U

  ,

  C

  G

  U

  –3′

  AND  

  5′–

  G

  C

  U

  ,

  A

  G

  A

  ,

  U

  A

  G

  –3′

• E.  

  5′–

  A

  C

  G

  ,

  A

  G

  A

  ,

  U

  G

  A

  –3′

  AND  

  5′–

  G

  C

  U

  ,

  A

  G

  A

  ,

  U

  A

  G

  –3′

• F.  

  5′–

  U

  G

  C

  ,

  U

  C

  U

  ,

  A

  C

  U

  –3′

  AND  

  5′–

  G

  A

  U

  ,

  A

  G

  A

  ,

  U

  C

  G

  –3′

Check Answer

 

RFLP Paternity Testing (EASY, 3 Males)

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The Question

Who is the father of the child?

Mother

Child

Male 1

Male 2

Male 3

Background

Restriction Fragment Length Polymorphism (RFLP) is a molecular biology technique used to distinguish between closely related DNA samples. It's commonly employed in paternity tests, among other applications.

Disclaimer

In actual diagnostic processes, the results of RFLP for confirming paternity are often cited to provide a 99.9% accuracy level or higher. However, no test is foolproof. Legal confirmation may involve additional procedures and evaluations to ensure the integrity and admissibility of the test.

Instructions

Use the provided DNA gel profile to determine paternity. Each band in the gel corresponds to a DNA fragment. Fragments are inherited; thus, the child's DNA will have overlapping fragments with the true father.

• A. Male #1
• B. Male #2
• C. Male #3

Check Answer

 

RFLP Paternity Testing (HARD, 9 Males)

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The Question

Who is the father of the child?

Mother

Child

Male 1

Male 2

Male 3

Male 4

Male 5

Male 6

Male 7

Male 8

Male 9

Background

Restriction Fragment Length Polymorphism (RFLP) is a molecular biology technique used to distinguish between closely related DNA samples. It's commonly employed in paternity tests, among other applications.

Disclaimer

In actual diagnostic processes, the results of RFLP for confirming paternity are often cited to provide a 99.9% accuracy level or higher. However, no test is foolproof. Legal confirmation may involve additional procedures and evaluations to ensure the integrity and admissibility of the test.

Instructions

Use the provided DNA gel profile to determine paternity. Each band in the gel corresponds to a DNA fragment. Fragments are inherited; thus, the child's DNA will have overlapping fragments with the true father.

• A. Male #1
• B. Male #2
• C. Male #3
• D. Male #4
• E. Male #5
• F. Male #6
• G. Male #7
• H. Male #8
• I. Male #9

Check Answer

 

RFLP Paternity Testing (MEDIUM, 5 Males)

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The Question

Who is the father of the child?

Mother

Child

Male 1

Male 2

Male 3

Male 4

Male 5

Background

Restriction Fragment Length Polymorphism (RFLP) is a molecular biology technique used to distinguish between closely related DNA samples. It's commonly employed in paternity tests, among other applications.

Disclaimer

In actual diagnostic processes, the results of RFLP for confirming paternity are often cited to provide a 99.9% accuracy level or higher. However, no test is foolproof. Legal confirmation may involve additional procedures and evaluations to ensure the integrity and admissibility of the test.

Instructions

Use the provided DNA gel profile to determine paternity. Each band in the gel corresponds to a DNA fragment. Fragments are inherited; thus, the child's DNA will have overlapping fragments with the true father.

• A. Male #1
• B. Male #2
• C. Male #3
• D. Male #4
• E. Male #5

Check Answer

 

RFLP Forensic DNA Analysis (EASY, 4 Suspects)

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The Question

Which suspect left blood at the crime scene?

Victim

Blood

Suspect #1

Suspect #2

Suspect #3

Suspect #4

Background

Restriction Fragment Length Polymorphism (RFLP) is a molecular biology method used to differentiate between closely related DNA samples. It is often used in forensic investigations.

Disclaimer

In adherence to the principles of due process, all individuals in this exercise shall be presumed innocent until proven guilty beyond a reasonable doubt in a court of law.

Instructions

Examine the provided DNA gel profile to identify which suspect is responsible for leaving the blood sample. Each band in the gel signifies a DNA fragment. The killer's DNA will match with the blood sample found at the crime scene.

• A. Suspect #1
• B. Suspect #2
• C. Suspect #3
• D. Suspect #4

Check Answer

 

RFLP Forensic DNA Analysis (HARD, 9 Suspects)

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The Question

Which suspect left blood at the crime scene?

Victim

Blood

Suspect #1

Suspect #2

Suspect #3

Suspect #4

Suspect #5

Suspect #6

Suspect #7

Suspect #8

Suspect #9

Background

Restriction Fragment Length Polymorphism (RFLP) is a molecular biology method used to differentiate between closely related DNA samples. It is often used in forensic investigations.

Disclaimer

In adherence to the principles of due process, all individuals in this exercise shall be presumed innocent until proven guilty beyond a reasonable doubt in a court of law.

Instructions

Examine the provided DNA gel profile to identify which suspect is responsible for leaving the blood sample. Each band in the gel signifies a DNA fragment. The killer's DNA will match with the blood sample found at the crime scene.

• A. Suspect #1
• B. Suspect #2
• C. Suspect #3
• D. Suspect #4
• E. Suspect #5
• F. Suspect #6
• G. Suspect #7
• H. Suspect #8
• I. Suspect #9

Check Answer

 

RFLP Forensic DNA Analysis (MEDIUM, 5 Suspects)

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The Question

Which suspect left blood at the crime scene?

Victim

Blood

Suspect #1

Suspect #2

Suspect #3

Suspect #4

Suspect #5

Background

Restriction Fragment Length Polymorphism (RFLP) is a molecular biology method used to differentiate between closely related DNA samples. It is often used in forensic investigations.

Disclaimer

In adherence to the principles of due process, all individuals in this exercise shall be presumed innocent until proven guilty beyond a reasonable doubt in a court of law.

Instructions

Examine the provided DNA gel profile to identify which suspect is responsible for leaving the blood sample. Each band in the gel signifies a DNA fragment. The killer's DNA will match with the blood sample found at the crime scene.

• A. Suspect #1
• B. Suspect #2
• C. Suspect #3
• D. Suspect #4
• E. Suspect #5

Check Answer