11: PCR and Electrophoresis

Students extract DNA, perform initial and nested PCR amplification of the GAPDH gene, run gel electrophoresis to separate DNA fragments, and interpret band patterns.

Gene Expression Fold Change from RT-qPCR Data

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	Untreated Cells		Cells Treated with Experimental Drug
	House Keeping Gene	Gene of Interest	House Keeping Gene	Gene of Interest
Well 1	19.0	26.2	17.8	23.4
Well 2	18.7	25.8	17.5	22.1
Well 3	18.6	25.8	17.7	22.2
mean Ct	18.8	25.9	17.7	22.6
ΔCt
ΔΔCt

Given the data in the table above calculate the fold change (2^|ΔΔCt|) value for the effect of the drug on the cells.
Note: answers need to be within 2% of the correct number to be correct.

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. 150 base pairs
• B. 400 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	2.63
1,000 base pairs	1000	2.26
2,000 base pairs	2000	1.90
3,000 base pairs	3000	1.69
5,000 base pairs	5000	1.42
10,000 base pairs	10000	1.06
Unknown	? ?	2.05

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 17% of the correct number to be correct.

Check Answer

 

Offspring HLA Genotypes (2 Markers, Color)

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HLA genotyping serves as a key component in the field of immunogenetics. It is used for understanding individual variations in immune responses. This molecular technique identifies unique alleles on paired chromosomes. It's particularly vital in organ transplantation for matching donors and recipients. Finding a correct match reduces the chance for graft rejection.
A mother has a HLA genotype of A8,B8 on one chromosome and A3,B7 on the other chromosome.
The father has a HLA genotype of A2,B6 on one chromosome and A7,B5 on the other chromosome.
Which one of the following combinations is a possible genotype for their offspring?

• A. B6,A7,B7,A8
• B. A2,B6,A8,B8
• C. A2,B6,A7,B5
• D. A3,B7,A8,B8
• E. A2,B5,B7,A8

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

  A

  C

  U

  ,

  C

  G

  U

  –3′

  AND  

  5′–

  G

  G

  G

  ,

  U

  C

  A

  –3′

• B.  

  5′–

  A

  C

  G

  ,

  A

  G

  U

  –3′

  AND  

  5′–

  U

  G

  A

  ,

  C

  C

  C

  –3′

• C.  

  5′–

  U

  G

  C

  ,

  U

  C

  A

  –3′

  AND  

  5′–

  G

  G

  G

  ,

  U

  C

  A

  –3′

• D.  

  5′–

  U

  G

  C

  ,

  U

  C

  A

  –3′

  AND  

  5′–

  A

  C

  U

  ,

  G

  G

  G

  –3′

• E.  

  5′–

  G

  G

  G

  ,

  U

  C

  A

  –3′

  AND  

  5′–

  U

  G

  A

  ,

  G

  C

  A

  –3′

• F.  

  5′–

  A

  C

  U

  ,

  G

  G

  G

  –3′

  AND  

  5′–

  A

  C

  U

  ,

  C

  G

  U

  –3′

Check Answer

 

Protein Molecular Weights from SDS-PAGE Band Positions

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Lane 1 shows a simulated Kaleidoscope-style pre-stained protein ladder.

Match each colored band to its molecular weight.

	Your Choice	Prompt
	Drop Your Choice Here	1.  colored band
	Drop Your Choice Here	2.  colored band
	Drop Your Choice Here	3.  colored band
	Drop Your Choice Here	4.  colored band

Drag one of the choices below:

• A. 25 kDa
• B. 150 kDa
• C. 10 kDa
• D. 75 kDa

Check Answer Reset Game

 

Protein Molecular Weight from an SDS-PAGE Ladder

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Below is a simulated SDS–PAGE gel.
Lane 1 contains a Kaleidoscope-style pre-stained protein ladder.
Lane 2 contains a single band labeled Protein X17.
The gel was run for too short a time (bands are compressed near the top).
Standard ladder reference (kDa):

			– 250
			– 150
			– 100
			– 75
			– 50
			– 37
			– 25
			– 20
			– 15
			– 10

Gel results:

Lane 1

Lane 2

Which protein (name and molecular weight) best matches Protein X17?
Use the ladder to estimate the band size. You do not need outside knowledge about the proteins.

• A. Transferrin (80.0 kDa)
• B. Estrogen Binding Protein (115.0 kDa)
• C. Aldolase (47.5 kDa)
• D. Green Fluorescent Protein (28.0 kDa)
• E. γ-Globulin (57.0 kDa)

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.

					HhaI				PstI								HhaI
	0		1		2		3		4		5		6		7		8		9		10		11		12

Two (2) distinct types of restriction enzyme recognition sites, HhaI and PstI, 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 HhaI only.

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

Check Answer Clear Selection

 

Nested PCR Primer Pair Selection (24 nt)

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

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

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

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

  A

  A

  ,

  G

  U

  C

  –3′

  AND  

  5′–

  C

  C

  G

  ,

  U

  C

  A

  –3′

• B.  

  5′–

  C

  C

  G

  ,

  U

  C

  A

  –3′

  AND  

  5′–

  C

  U

  G

  ,

  A

  A

  G

  –3′

• C.  

  5′–

  G

  A

  C

  ,

  U

  U

  C

  –3′

  AND  

  5′–

  C

  U

  G

  ,

  A

  A

  G

  –3′

• D.  

  5′–

  A

  C

  U

  ,

  G

  C

  C

  –3′

  AND  

  5′–

  C

  U

  G

  ,

  A

  A

  G

  –3′

• E.  

  5′–

  A

  C

  U

  ,

  G

  C

  C

  –3′

  AND  

  5′–

  G

  A

  A

  ,

  G

  U

  C

  –3′

• F.  

  5′–

  C

  C

  G

  ,

  U

  C

  A

  –3′

  AND  

  5′–

  A

  C

  U

  ,

  G

  C

  C

  –3′

Check Answer

 

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

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5′–

A

G

C

,

G

T

G

,

G

A

C

,

T

C

T

,

C

T

G

,

T

C

C

,

C

A

A

,

G

G

T

,

A

G

G

,

C

A

G

,

C

G

A

,

T

A

G

–3′

3′–

T

C

G

,

C

A

C

,

C

T

G

,

A

G

A

,

G

A

C

,

A

G

G

,

G

T

T

,

C

C

A

,

T

C

C

,

G

T

C

,

G

C

T

,

A

T

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′–

  G

  U

  C

  ,

  C

  A

  C

  ,

  G

  C

  U

  –3′

  AND  

  5′–

  U

  C

  G

  ,

  C

  A

  C

  ,

  C

  U

  G

  –3′

• B.  

  5′–

  C

  A

  G

  ,

  G

  U

  G

  ,

  C

  G

  A

  –3′

  AND  

  5′–

  G

  U

  C

  ,

  G

  C

  U

  ,

  A

  U

  C

  –3′

• C.  

  5′–

  A

  G

  C

  ,

  G

  U

  G

  ,

  G

  A

  C

  –3′

  AND  

  5′–

  G

  U

  C

  ,

  G

  C

  U

  ,

  A

  U

  C

  –3′

• D.  

  5′–

  C

  A

  G

  ,

  C

  G

  A

  ,

  U

  A

  G

  –3′

  AND  

  5′–

  C

  A

  G

  ,

  G

  U

  G

  ,

  C

  G

  A

  –3′

• E.  

  5′–

  C

  U

  A

  ,

  U

  C

  G

  ,

  C

  U

  G

  –3′

  AND  

  5′–

  C

  A

  G

  ,

  G

  U

  G

  ,

  C

  G

  A

  –3′

• F.  

  5′–

  A

  G

  C

  ,

  G

  U

  G

  ,

  G

  A

  C

  –3′

  AND  

  5′–

  C

  U

  A

  ,

  U

  C

  G

  ,

  C

  U

  G

  –3′

Check Answer

 

Protein Molecular Weight from SDS-PAGE Migration

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Gel Migration Problem

In this task, data from an SDS-PAGE experiment, where proteins are separated based on molecular weight, is provided. The gel results table below shows some standard proteins with known molecular weights and one unknown protein.

Protein Name	Molecular Weight (kDa)	Migration Distance (cm)
Cytochrome c (Cyt)	13.0	3.45
Myelin Basic Protein (MBP)	18.0	3.19
Serine Protease (Ser)	22.0	3.03
Green Fluorescent Protein (GFP)	28.0	2.83
Lipase (Lip)	40.0	2.55
Aldolase (Aldo)	47.5	2.41
Serum Albumin (Alb)	66.2	2.15
Unknown	?	3.37

Estimate the molecular weight of the unknown protein by comparing its gel migration distance with those of the standards.

• A. 14 kDa
• B. 20 kDa
• C. 24 kDa
• D. 31 kDa
• E. 44 kDa

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