Recombinant DNA Technology and Biotechnology Review Questions with Complete Solutions Rated
A+
What are restriction endonucleases? - Answers enzymes that cleave very specific DNA sequences;
restriction sequences are generally short (usually 4-8 bp)
What type of sequences are usually recognized by restriction endonucleases? What is special about
these sequences? - Answers palindromes--read the same 5'-->3'
coding and template strand are the same
What are the two possibilities when restriction enzymes cleave DNA? - Answers sticky (staggered or
cohesive end) or blunt ends; 3' OH group and 5' phosphate are attached after cleavage (important for
ligation)
Restriction enzymes are generally named for what? - Answers the organism that they were derived from
What is a restriction site? - Answers DNA sequence that can be cleaved by a restriction enzyme
What can be said about restriction enzymes that recognize larger sequences in terms of how often they
cut? - Answers cut less frequently; in theory--4 base recognition sequence would be 1 in 256 bp; 6 base
recognition sequence would be 1 in 2500 bp
What is recombinant DNA? What enzyme makes this possible and what does the enzyme do? - Answers
fragments of DNA that are pasted together to make hybrid molecules; very easy with sticky ends; DNA
ligase creates the phosphodiester bonds
What does DNA cloning involve? - Answers inserting a restriction fragment into a cloning vector; a
vector can then be replicated in host cells (usually bacteria, sometimes yeast or other cells); DNA is now
cloned and amplified; Recombinant DNA amplification
What are vectors? What are the requirements for a vector? - Answers molecules of DNA that can accept
fragments of foreign DNA; must be capable of autonomous replication of cell; must have at least one
restriction site for foreign DNA insertion; must carry at least one gene for selection (usually antibiotic
resistance)
What are the most common vectors? - Answers prokaryotic plasmids; other vectors--phages, yeast,
plasmids and yeast artificial chromosomes (YACs) and mammalian viruses (example--retroviruses)
What are the two types of DNA libraries? - Answers genomic DNA libraries and cDNA libraries
What is a genomic DNA library? - Answers entire genome is chopped up with restriction enzymes,
cloned into vectors, and used to transform bacteria; each transformed bacteria containing a plasmid
may contain a different segment of the genome (thousands are collected to assure the whole genome is
represented); the collection contains all sequences in the genome, including coding regions as well as
introns and other intervening sequences, promoters, etc.
, What is a cDNA library? - Answers complementary DNA; cDNA is generated using isolated mRNA from a
particular cell or tissue type; mRNA is reverse transcribed (by reverse transcriptase) and the second
strand is synthesized using a DNA polymerase; cDNA is ligated into a vector, used to transform bacteria
and 1000s of clones are collected; Ideally, cDNA library will contain sequences representing all mRNAs
present in the cell or tissue type at the time the mRNA was collected
What does a cDNA library allow? What type of information does it use? - Answers allows one to see
what genes were being expressed in a particular cell or tissue type; only contains mRNA sequences--no
introns, promoters, etc.
What can be done with DNA from a cDNA library? - Answers can be coned into an expression vector for
production of proteins; bacterial expression vector--promoter, Shine-Dalgarno sequence and cDNA are
used to transform expression bacteria strains; other vector types as well (yeast, mammalian)
What is DNA sequencing? - Answers technique used to determine the exact sequence of a cloned or PCR
amplified stretch of DNA
What does melting accomplish in DNA sequencing? - Answers generates single stranded template
What are the major components of DNA sequencing reactions? - Answers DNA, dNTPs, DNA primer and
DNA polymerase; sample split into 4 tubes
What does each tube contain for DNA sequencing? What step follows after? - Answers small amount of
specific dideoxyribonucleotide: ddCTP, ddATP, ddGTP, and ddTTP; conduct PCR sequencing reaction and
then apply samples to an agarose gel for electrophoresis
What are probes used for? - Answers to identify DNA fragments; probe is a ssDNA molecule that is
labeled using radioactivity that can be hybridized as ssDNA that is complementary
What are the steps of hybridization? - Answers target DNA is made ss (heat, chemicals); target is
immobilized on a solid support (nitrocellulose membrane) so it cannot re-anneal with its original
complementary strand; ssDNA coated membrane is exposed to probe; if complementary sequence is
present, probe will bind the immobilized ssDNA and can be identified via autoradiography
Describe smaller probes. - Answers chemically synthesized oligonucleotides (2-30 bases); same way
synthetic primers are made; smaller probes can be very specific i.e. identify a single base mutation in a
sequence
Describe larger probes. - Answers made one of several molecular biology techniques ( reverse
transcription, PCR, etc); much less specific--can be used to identify similar genes in different organisms
or the same gene in different individuals that may not be exactly the same in sequence
What is Southern blotting? - Answers analysis of DNA; DNA is isolated and subject to restriction
digestion; digested DNA is then subjected to gel electrophoresis; DNA is denatured and blotted
(immobilized onto a membrane); blot (membrane is probed)
A+
What are restriction endonucleases? - Answers enzymes that cleave very specific DNA sequences;
restriction sequences are generally short (usually 4-8 bp)
What type of sequences are usually recognized by restriction endonucleases? What is special about
these sequences? - Answers palindromes--read the same 5'-->3'
coding and template strand are the same
What are the two possibilities when restriction enzymes cleave DNA? - Answers sticky (staggered or
cohesive end) or blunt ends; 3' OH group and 5' phosphate are attached after cleavage (important for
ligation)
Restriction enzymes are generally named for what? - Answers the organism that they were derived from
What is a restriction site? - Answers DNA sequence that can be cleaved by a restriction enzyme
What can be said about restriction enzymes that recognize larger sequences in terms of how often they
cut? - Answers cut less frequently; in theory--4 base recognition sequence would be 1 in 256 bp; 6 base
recognition sequence would be 1 in 2500 bp
What is recombinant DNA? What enzyme makes this possible and what does the enzyme do? - Answers
fragments of DNA that are pasted together to make hybrid molecules; very easy with sticky ends; DNA
ligase creates the phosphodiester bonds
What does DNA cloning involve? - Answers inserting a restriction fragment into a cloning vector; a
vector can then be replicated in host cells (usually bacteria, sometimes yeast or other cells); DNA is now
cloned and amplified; Recombinant DNA amplification
What are vectors? What are the requirements for a vector? - Answers molecules of DNA that can accept
fragments of foreign DNA; must be capable of autonomous replication of cell; must have at least one
restriction site for foreign DNA insertion; must carry at least one gene for selection (usually antibiotic
resistance)
What are the most common vectors? - Answers prokaryotic plasmids; other vectors--phages, yeast,
plasmids and yeast artificial chromosomes (YACs) and mammalian viruses (example--retroviruses)
What are the two types of DNA libraries? - Answers genomic DNA libraries and cDNA libraries
What is a genomic DNA library? - Answers entire genome is chopped up with restriction enzymes,
cloned into vectors, and used to transform bacteria; each transformed bacteria containing a plasmid
may contain a different segment of the genome (thousands are collected to assure the whole genome is
represented); the collection contains all sequences in the genome, including coding regions as well as
introns and other intervening sequences, promoters, etc.
, What is a cDNA library? - Answers complementary DNA; cDNA is generated using isolated mRNA from a
particular cell or tissue type; mRNA is reverse transcribed (by reverse transcriptase) and the second
strand is synthesized using a DNA polymerase; cDNA is ligated into a vector, used to transform bacteria
and 1000s of clones are collected; Ideally, cDNA library will contain sequences representing all mRNAs
present in the cell or tissue type at the time the mRNA was collected
What does a cDNA library allow? What type of information does it use? - Answers allows one to see
what genes were being expressed in a particular cell or tissue type; only contains mRNA sequences--no
introns, promoters, etc.
What can be done with DNA from a cDNA library? - Answers can be coned into an expression vector for
production of proteins; bacterial expression vector--promoter, Shine-Dalgarno sequence and cDNA are
used to transform expression bacteria strains; other vector types as well (yeast, mammalian)
What is DNA sequencing? - Answers technique used to determine the exact sequence of a cloned or PCR
amplified stretch of DNA
What does melting accomplish in DNA sequencing? - Answers generates single stranded template
What are the major components of DNA sequencing reactions? - Answers DNA, dNTPs, DNA primer and
DNA polymerase; sample split into 4 tubes
What does each tube contain for DNA sequencing? What step follows after? - Answers small amount of
specific dideoxyribonucleotide: ddCTP, ddATP, ddGTP, and ddTTP; conduct PCR sequencing reaction and
then apply samples to an agarose gel for electrophoresis
What are probes used for? - Answers to identify DNA fragments; probe is a ssDNA molecule that is
labeled using radioactivity that can be hybridized as ssDNA that is complementary
What are the steps of hybridization? - Answers target DNA is made ss (heat, chemicals); target is
immobilized on a solid support (nitrocellulose membrane) so it cannot re-anneal with its original
complementary strand; ssDNA coated membrane is exposed to probe; if complementary sequence is
present, probe will bind the immobilized ssDNA and can be identified via autoradiography
Describe smaller probes. - Answers chemically synthesized oligonucleotides (2-30 bases); same way
synthetic primers are made; smaller probes can be very specific i.e. identify a single base mutation in a
sequence
Describe larger probes. - Answers made one of several molecular biology techniques ( reverse
transcription, PCR, etc); much less specific--can be used to identify similar genes in different organisms
or the same gene in different individuals that may not be exactly the same in sequence
What is Southern blotting? - Answers analysis of DNA; DNA is isolated and subject to restriction
digestion; digested DNA is then subjected to gel electrophoresis; DNA is denatured and blotted
(immobilized onto a membrane); blot (membrane is probed)
