October 2012
NAME: ____________________
Biotechnology Summary
Matching – Use the following words to fill in the blanks below: restriction enzyme, gel electrophoresis, transformation,
polymerase chain reaction, restriction fragment length polymorphism, DNA sequencing, DNA chip/microarray
a technique that exploits variations in homologous DNA sequences
the process of reading the nucleotide bases in a DNA molecule
genetic alteration of a cell resulting from the direct uptake, incorporation
and expression of exogenous genetic material (exogenous DNA) from its
surroundings and taken up through the cell membrane(s)
a biochemical technology in molecular biology to amplify a single or a few
copies of a piece of DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA sequence
a method used in clinical chemistry to separate proteins by charge and or
size
used to measure the expression levels of large numbers of genes
simultaneously or to genotype multiple regions of a genome
thought to have evolved to provide a defense mechanism against invading
viruses
Restriction Enzymes, Methylases & DNA Ligase
____
____
____
____
1. Restriction endonuclease is selected for use based on
a. the repetition of one sequence.
b. sequence position.
c. relative overhand.
d. sequence length.
e. a specific nucleotide sequence.
2. Some restriction endonucleases form blunt ends. This allows
a. only one product is formed.
b. any two pairs can be combined.
c. extremely pure products.
d. A-T pairs to form T-A pairs.
e. only one form of combination.
3. One step in producing recombinant DNA is the use of DNA ligase. This is intended to
a. open the GAATTC site.
b. seal the DNA breaks.
c. form a plasmid.
d. help match sticky ends.
e. make the new DNA a loop.
4. What is true of restriction enzymes?
a. They normally occur in prokaryotes.
b. They attach to DNA at specific recognition sites.
c. They break hydrogen bonds between nitrogenous bases.
d. All of the above.
____
5. What is true of restriction fragments?
a. They can end with unpaired DNA nucleotides.
b. They are segments of DNA that have been cut by restriction enzymes.
c. They can end with paired DNA nucleotides.
d. All of the above.
____
6. What is true of the recognition sites for restriction enzymes?
a. They are palindromic DNA sequences.
b. They are found only on the leading strand.
c. They are found only in prokaryotic cells.
d. They are found only on the coding strand.
____
7. Restriction enzymes are crucial to recombinant DNA technology because they
a. cut DNA at either end of a gene.
b. cut DNA at specific and predictable sequences of bases.
c. tag DNA so that individual fragments can be identified.
d. cut DNA, leaving unpaired lengths of bases that have a charge.
8. What bonds can restriction enzymes break?
a. covalent bonds only
b. hydrogen bonds only
c. hydrogen bonds and ionic bonds
d. covalent bonds and hydrogen bonds
____
____
9. The recognition site of the SmaI restriction enzyme is shown below.
SmaI
5'-GGGCCC-3'
3'-CCCGGG-5'
The restriction fragments would
a. produce blunt ends with A-T base pairs.
b. produce sticky ends with A-T base pairs.
c. produce sticky ends with G-C base pairs.
d. produce blunt ends with G-C base pairs.
____ 10. The recognition sites of five restriction enzymes are shown. Which of these enzymes would make
cuts in the following DNA fragment? (Hint: The complementary DNA strand is needed to answer
this question.)
5'-GCAAGCTTGCTGGATCCTTACCCGGA-3'
Recognition Sites:
5'-GAATTC-3'
EcoRI
3'-CTTAAG-5'
SmaI
5'-GGGCCC-3'
3'-CCCGGG-5'
BamHI
5'-GGATCC-3'
3'-CCTAGG-5'
AluI
5'-AGCT-3'
3'-TCGA-5'
HindIII
5'-AAGCTT-3'
3'-TTCGAA-5'
a.
b.
c.
d.
SmaI and HindIII
HindIII and BamHI
EcoRI and HindIII
EcoRI and AluI
____ 11. The EcoRI restriction enzyme can break only two hydrogen bonds and has the recognition site
shown.
5'-GAATTC-3'
EcoRI
3'-CTTAAG-5'
Based on this information, what would you expect to see in the resulting restriction fragments?
a. blunt ends that still have A-T base pairs
b. sticky ends that still have A-T base pairs
c. sticky ends that still have G-C base pairs
d. blunt ends that still have G-C base pairs
____ 12. The recognition sites of five restriction enzymes are shown. You need to remove the underlined
sequence for further study. Which enzymes would you use to isolate the DNA fragment? (Hint: The
complementary DNA strand is needed for this question.)
5'-GCAAGGGCCCTAGCTGCAGAAGCTTACCCGGA-3'
Recognition Sites:
5'-GAATTC-3'
3'-CTTAAG-5'
EcoRI
SmaI
5'-GGGCCC-3'
3'-CCCGGG-5'
BamHI
5'-GGATCC-3'
3'-CCTAGG-5'
AluI
5'-AGCT-3'
3'-TCGA-5'
HindIII
5'-AAGCTT-3'
3'-TTCGAA-5'
a.
b.
c.
d.
SmaI and AluI
HindIII and SmaI
EcoRI and AluI
AluI and HindIII
____ 13. The restriction enzyme AluI leaves blunt ends on DNA fragments after digestion. These blunt ends
a. require a polymerase enzyme to bond them to other blunt ends.
b. are fully base paired.
c. form hydrogen bonds with the blunt ends of other fragments produced by AluI.
d. have only one unpaired base.
____ 14. The ends of the DNA fragments shown have been produced by digestion of a single sequence of
DNA using a number of restriction endonucleases.
1.
5'A
3'
2. 5'G
3'
3'TTCGA5'
3'CAGCT5'
3.
5'AATTC3'
3'
G5’
4.
5'TCGAC
3'
G5'3'
5.
5'GGG 3'
3'CCC 5'
Which of these ends are capable of annealing and being joined by DNA ligase?
A
3 and 4
B
1 and 2
C
1 and 5
D
2 and 4
15. Recombinant DNA requires restriction endonuclease to make restriction fragments. Some of these
fragments have sticky ends. Some of the fragments have blunt ends.
Explain the advantage of each type of end.
16. What role does restriction endonuclease play in the formation of recombinant DNA?
Gel Electrophoresis
1. What is the role of the electric source in gel electrophoresis?
2. How does ethidium bromide contribute to gel electrophoresis?
3. What is a lane in the process of gel electrophoresis?
Plasmids and Transformation
1. Recombinant DNA developed in bacteria. Explain this.
2. A plasmid is digested using the restriction enzymes SmaI and XhoI, with the following results. Use this data
to construct a plasmid map.
SmaI
100 bp
400 bp
500 bp
XhoI
400 bp
600 bp
SmaI and XhoI
100 bp
200 bp
300 bp
400 bp
Polymerase Chain Reaction
1. How are the different temperature changes used in PCR technology?
Restriction Fragment Length Polymorphism
1. Why are restriction enzymes and gel electrophoresis used in RFLP?
2. Although now largely obsolete due to the rise of inexpensive DNA sequencing technologies, what was RFLP
analysis used for in the past? Give three uses.
DNA Sequencing
1. What is dideoxy chain termination?
2. When running a gel during a Sanger sequencing, the following results were obtained:
Find the DNA sequence that would be the correct interpretation of this sequence:
3. Follow the procedure below to answer this question.
a) Remember that the tube title indicates which respective dideoxynucleotide each tube
contains.
b) The sequence you have been given represents the single-stranded DNA fragment that is to
be sequenced.
Tube G
5’ G
A
C
A
T
T
3’
G
C
C
A
T
A
G
A
C
A
T
T
A
G
C
3’
A A T C G
5’
c) You have been given the 5 nucleotide primer for the template strand (bold).
d) For your given tube, “synthesize” the complementary strand and terminate with a
dideoxynucleotide each time a nucleotide is reached that is complementary to that reaction
tube ddNTP.
e) Count the number of fragments and length of each fragment that was synthesized. Write
answer on answer page.
DNA Chip/Microarray
1. What is a DNA microarray?
2. What are probes in terms of a microarray?
Final Practice Question
When running a gel during a Sanger sequencing, the following results were obtained:
(a) Find the DNA sequence that would be the correct interpretation of this sequence.
(b) Find the mRNA sequence that would be the correct interpretation of this sequence.
(c) Use the genetic code to find the polypeptide sequence that would be the correct interpretation of
this sequence. (Hint: remember to read DNA and mRNA in a 5' to 3' direction)
October 2012
NAME: I AM RIGHT
Biotechnology Summary
Matching – Use the following words to fill in the blanks below: restriction enzyme, gel electrophoresis, transformation,
polymerase chain reaction, restriction fragment length polymorphism, DNA sequencing, DNA chip/microarray
restriction fragment length
polymorphism
DNA sequencing
transformation
polymerase chain reaction
gel electrophoresis
DNA chip/microarray
Restriction enzyme
a technique that exploits variations in homologous DNA sequences
the process of reading the nucleotide bases in a DNA molecule
genetic alteration of a cell resulting from the direct uptake, incorporation
and expression of exogenous genetic material (exogenous DNA) from its
surroundings and taken up through the cell membrane(s)
a biochemical technology in molecular biology to amplify a single or a few
copies of a piece of DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA sequence
a method used in clinical chemistry to separate proteins by charge and or
size
used to measure the expression levels of large numbers of genes
simultaneously or to genotype multiple regions of a genome
thought to have evolved to provide a defense mechanism against invading
viruses
Restriction Enzymes, Methylases & DNA Ligase
____
____
____
____
1. Restriction endonuclease is selected for use based on
a. the repetition of one sequence.
b. sequence position.
c. relative overhand.
d. sequence length.
e. a specific nucleotide sequence.
2. Some restriction endonucleases form blunt ends. This allows
a. only one product is formed.
b. any two pairs can be combined.
c. extremely pure products.
d. A-T pairs to form T-A pairs.
e. only one form of combination.
3. One step in producing recombinant DNA is the use of DNA ligase. This is intended to
a. open the GAATTC site.
b. seal the DNA breaks.
c. form a plasmid.
d. help match sticky ends.
e. make the new DNA a loop.
4. What is true of restriction enzymes?
a. They normally occur in prokaryotes.
b. They attach to DNA at specific recognition sites.
c. They break hydrogen bonds between nitrogenous bases.
d. All of the above.
____
5. What is true of restriction fragments?
a. They can end with unpaired DNA nucleotides.
b. They are segments of DNA that have been cut by restriction enzymes.
c. They can end with paired DNA nucleotides.
d. All of the above.
____
6. What is true of the recognition sites for restriction enzymes?
a. They are palindromic DNA sequences.
b. They are found only on the leading strand.
c. They are found only in prokaryotic cells.
d. They are found only on the coding strand.
____
7. Restriction enzymes are crucial to recombinant DNA technology because they
a. cut DNA at either end of a gene.
b. cut DNA at specific and predictable sequences of bases.
c. tag DNA so that individual fragments can be identified.
d. cut DNA, leaving unpaired lengths of bases that have a charge.
8. What bonds can restriction enzymes break?
a. covalent bonds only
b. hydrogen bonds only
c. hydrogen bonds and ionic bonds
d. covalent bonds and hydrogen bonds
____
____
9. The recognition site of the SmaI restriction enzyme is shown below.
SmaI
5'-GGGCCC-3'
3'-CCCGGG-5'
The restriction fragments would
a. produce blunt ends with A-T base pairs.
b. produce sticky ends with A-T base pairs.
c. produce sticky ends with G-C base pairs.
d. produce blunt ends with G-C base pairs.
____ 10. The recognition sites of five restriction enzymes are shown. Which of these enzymes would make
cuts in the following DNA fragment? (Hint: The complementary DNA strand is needed to answer
this question.)
5'-GCAAGCTTGCTGGATCCTTACCCGGA-3'
Recognition Sites:
5'-GAATTC-3'
EcoRI
3'-CTTAAG-5'
SmaI
5'-GGGCCC-3'
3'-CCCGGG-5'
BamHI
5'-GGATCC-3'
3'-CCTAGG-5'
AluI
5'-AGCT-3'
3'-TCGA-5'
HindIII
5'-AAGCTT-3'
3'-TTCGAA-5'
a.
b.
c.
d.
SmaI and HindIII
HindIII and BamHI
EcoRI and HindIII
EcoRI and AluI
____ 11. The EcoRI restriction enzyme can break only two hydrogen bonds and has the recognition site
shown.
5'-GAATTC-3'
EcoRI
3'-CTTAAG-5'
Based on this information, what would you expect to see in the resulting restriction fragments?
a. blunt ends that still have A-T base pairs
b. sticky ends that still have A-T base pairs
c. sticky ends that still have G-C base pairs
d. blunt ends that still have G-C base pairs
____ 12. The recognition sites of five restriction enzymes are shown. You need to remove the underlined
sequence for further study. Which enzymes would you use to isolate the DNA fragment? (Hint: The
complementary DNA strand is needed for this question.)
5'-GCAAGGGCCCTAGCTGCAGAAGCTTACCCGGA-3'
Recognition Sites:
5'-GAATTC-3'
3'-CTTAAG-5'
EcoRI
SmaI
5'-GGGCCC-3'
3'-CCCGGG-5'
BamHI
5'-GGATCC-3'
3'-CCTAGG-5'
AluI
5'-AGCT-3'
3'-TCGA-5'
HindIII
5'-AAGCTT-3'
3'-TTCGAA-5'
a.
b.
c.
d.
SmaI and AluI
HindIII and SmaI
EcoRI and AluI
AluI and HindIII
____ 13. The restriction enzyme AluI leaves blunt ends on DNA fragments after digestion. These blunt ends
a. require a polymerase enzyme to bond them to other blunt ends.
b. are fully base paired.
c. form hydrogen bonds with the blunt ends of other fragments produced by AluI.
d. have only one unpaired base.
____ 14. The ends of the DNA fragments shown have been produced by digestion of a single sequence of
DNA using a number of restriction endonucleases.
1.
5'A
3'
2. 5'G
3'
3'TTCGA5'
3'CAGCT5'
3.
5'AATTC3'
3'
G5’
4.
5'TCGAC
3'
G5'3'
5.
5'GGG 3'
3'CCC 5'
Which of these ends are capable of annealing and being joined by DNA ligase?
A
3 and 4
B
1 and 2
C
1 and 5
D
2 and 4
Recombinant DNA requires restriction endonuclease to make restriction fragments. Some of these fragments
have sticky ends. Some of the fragments have blunt ends.
Explain the advantage of each type of end.
ANS:
Sticky ends - one nucleotide strand has some unattached nucleotides that can bind with a specific
sequence. This allows for sequence specificity
What role does restriction endonuclease play in the formation of recombinant DNA?
ANS:
Restriction endonucleases target and break specific sequences of interior DNA strands
Gel Electrophoresis
What is the role of the electric source in gel electrophoresis?
ANS:
The electric source provides two polar ends (negative charge and positive charge). DNA fragments
move toward the positive end according to size.
How does ethidium bromide contribute to gel electrophoresis?
ANS:
Ethidium bromide associates with the DNA to fluoresces under UV.
What is a lane in the process of gel electrophoresis?
ANS:
A lane is a space in the gel where a sample is placed.
Plasmids and Transformation
Recombinant DNA developed in bacteria. Explain this.
ANS:
Bacteria have the capacity of transformation. A plasmid can be produced and then incorporated into
a bacterium through transformation.
A plasmid is digested using the restriction enzymes SmaI and XhoI, with the following results. Use this data to
construct a plasmid map.
SmaI
100 bp
400 bp
500 bp
XhoI
400 bp
600 bp
SmaI and XhoI
100 bp
200 bp
300 bp
400 bp
ANS:
Polymerase Chain Reaction
How are the different temperature changes used in PCR technology?
ANS:
The high temperature (95C) breaks the hydrogen bonds that connect the double stranded DNA, or
denaturation. The cooler temperature (55C) allows the strands to combine with the added primers,
or primer annealing. The middle temperature (72C) allows Taq polymerase to add nucletides, or
DNA synthesis.
Restriction Fragment Length Polymorphism
Why are restriction enzymes and gel electrophoresis used in RFLP?
Ans:
In RFLP analysis, the DNA sample is broken into pieces (digested) by restriction enzymes and the resulting
restriction fragments are separated according to their lengths by gel electrophoresis.
Although now largely obsolete due to the rise of inexpensive DNA sequencing technologies, what was RFLP
analysis used for in the past? Give three uses.
Ans:
RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application. In
addition to genetic fingerprinting, RFLP was an important tool in genome mapping, localization of genes for
genetic disorders, determination of risk for disease, and paternity testing.
DNA Sequencing
What is dideoxy chain termination?
ANS:
Dideoxy nucleotides are missing the -OH group at the 3’ end. This will not allow new nucleotides to
be added to the strand, so the nucleotide is terminated.
When running a gel during a Sanger sequencing, the following results were obtained:
Find the DNA sequence that would be the correct interpretation of this sequence:
ANS:
5' GTAGCCTATGAT 3'
Follow the procedure below to answer this question.
a) Remember that the tube title indicates which respective dideoxynucleotide each tube
contains.
b) The sequence you have been given represents the single-stranded DNA fragment that is to
be sequenced.
Tube G
5’ G
A
C
A
T
T
3’
G
C
C
A
T
A
G
A
C
A
T
T
A
G
C
3’
A A T C G
5’
c) You have been given the 5 nucleotide primer for the template strand (bold).
d) For your given tube, “synthesize” the complementary strand and terminate with a
dideoxynucleotide each time a nucleotide is reached that is complementary to that reaction
tube ddNTP.
e) Count the number of fragments and length of each fragment that was synthesized. Write
answer on answer page.
Ans:
4 fragments
Fragment 1(length 7)
Fragment 2 (13)
Fragment 3 (14)
Fragment 4 (length 19)
DNA Chip/Microarray
What is a DNA microarray?
Ans:
A DNA microarray (also commonly known as DNA chip) is a collection of microscopic DNA spots attached to a solid
surface.
What are probes in terms of a microarray?
Ans:
Each DNA spot contains a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section
of a gene that are used to hybridize a cDNA.
Final Practice Question
When running a gel during a Sanger sequencing, the following results were obtained:
(a) Find the DNA sequence that would be the correct interpretation of this sequence.
(b) Find the mRNA sequence that would be the correct interpretation of this sequence.
(c) Use the genetic code to find the polypeptide sequence that would be the correct interpretation of
this sequence. (Hint: remember to read DNA and mRNA in a 5' to 3' direction)
ANS:
(a) DNA 5' GTAGCCTATGAT 3'
(b) mRNA 3' CAU CGG AUA CUA 5'
(c) Ile-Ile-Gly-Tyr