Chapter 20: Recombinant DNA Technology, Mastering Genetics

Question 1

What is the function of restriction endonucleases in bacteria?

Answer 1

They provide a defense mechanism against infection by viruses.

Restriction endonucleases recognize and degrade viral DNA, thus preventing viral infections.

Question 2

TRUE or FALSE?

Restriction endonucleases cut DNA at specific recognition sequences and then bond two strands covalently with the same “sticky ends.”

Answer 2

False

Restriction endonucleases cut DNA at specific sequences, but DNA ligase must be used to bond two strands covalently with the same “sticky ends.”

Question 3

BamHI cuts the sequence 5′ G|GATCC 3′. Which of the following sequences would not be recognized by this enzyme?

Answer 3

BamHI cuts the sequence 5′ G|GATCC 3′. Which of the following sequences would not be recognized by this enzyme?

This sequence does not contain the BamHI recognition site.

Question 4

Which of the following DNA sequences is one strand of a restriction enzyme recognition sequence?

5’ AAACCC 3’
5’ GGGTTT 3’
5’ GGGGGG 3’
5’ GGATCC 3’

Answer 4

5’ GGATCC 3’

The 5’ → 3’ sequence of the complementary strand would be the same as the 5’ → 3’ sequence of this strand; i.e., this sequence is symmetrical about the midpoint.

Question 5

X-Gal is included in the growth medium on which cells transformed with bacterial plasmids are grown. The reason X-Gal is included is to _______.

Answer 5

identify bacteria that contain a recombinant plasmid

Colonies produced from cells containing a recombinant plasmid are white, whereas colonies from cells containing a nonrecombinant plasmid are blue.

Question 6

TRUE or FALSE?

Within a six-base DNA recognition sequence, an enzyme that cuts between the 3rd and 4th bases from the 5’ end will generate blunt ends.

Answer 6

True

Question 7

Restriction endonucleases are especially useful if they generate “sticky” ends. What makes an end sticky?

Answer 7

single-stranded complementary tails

Question 8

Assume that a given plasmid vector to be used in a cloning experiment contains 4000 base pairs of DNA. Assume also that the restriction endonuclease Cuj cuts this plasmid at the following sites (starting from an arbitrary zero point): 1000, 1500, and 3000.

Given complete digestion of the plasmid with the endonuclease so that only linear fragments are produced, what sizes of DNA are expected?

Answer 8

500 bp, 1500 bp, and 2000 bp

Question 9

TRUE or FALSE?

To isolate a bacterium with a plasmid that carries a desired DNA fragment cloned within the ampicillin resistance gene, we should grow bacteria in a medium that contains ampicillin.

Answer 9

False

Question 10

TRUE or FALSE?

In general, the main goal of cloning is to include as many different genes as possible in a single cloning vector.

Answer 10

False

Question 11

TRUE or FALSE?

A common term for a plasmid or other DNA element that serves as a cloning vehicle is vector.

Answer 11

True

Question 12

Following are 4 processes common to most cloning experiments:

A) transforming bacteria
B) plating bacteria on selective medium
C) cutting DNA with restriction endonucleases
D) ligating DNA fragments

Place components of this list in the order in which they would most likely occur during a cloning experiment.

Answer 12

1. Cutting DNA with restriction endonucleases.
2. Ligating DNA fragments.
3. Transforming bacteria.
4. Plating bacteria on selective medium.

Question 13

Some vectors such as pUC18 and others of the pUC series contain a large number of restriction enzyme sites clustered in one region. Which term is given to this advantageous arrangement of restriction sites?

Answer 13

multiple cloning site

Question 14

What term is used to refer to the process in which DNA can be introduced into host bacterial cells?

Answer 14

transformation

Question 15

Assume that one conducted a typical cloning experiment using a typical plasmid, transformed an appropriate host bacterial strain, and plated the bacteria on an appropriate X-gal medium. Blue and white colonies appeared. Which of the two types of colonies, blue or white, would most likely contain the recombinant plasmid?

Answer 15

white colonies

Question 16

TRUE or FALSE?

Phage λ can carry larger DNA fragments than plasmids.

Answer 16

True

Phage vectors can carry DNA fragments of about 20 kb, whereas plasmids can only carry DNA of less than 15 kb.

Question 17

Which of the following elements is not found in a plasmid?

Answer 17

Lambda arms

Lambda arms are regions that flank the inserted foreign DNA in phage λ vectors.

Question 18

A DNA fragment is introduced into the lacZ gene of a plasmid, which also contains a tetracycline resistance gene. What is the appearance of bacteria transformed with this plasmid if they are spread on plates containing tetracycline and Xgal?

Answer 18

White colonies that are resistant to tetracycline

The presence of blue colonies means that the plasmid taken up by these bacteria is recombinant, since the lacZ gene was disrupted.

Question 19

Why are filters overlaid with X‑ray film when screening a cDNA library?

Answer 19

To visualize probe hybridization events

The X‑ray film allows probe hybridization events on the filter to be visualized if a radioactive probe is used.

Question 20

TRUE or FALSE?

Chromosome walking is used mainly to isolate a gene when the sequence of the gene is known.

Answer 20

False

Chromosome walking is used mainly when the gene of interest has not yet been cloned but its approximate location on a chromosome is known.

Question 21

One of the primary reasons for generating a large number of clones in a eukaryotic genomic library is that ________.

Answer 21

each vector can take up only a relatively small fraction of the eukaryotic DNA

Question 22

Which type of DNA library represents the genes expressed by a given cell at a certain time?

Answer 22

cDNA

cDNA libraries represent expressed genes from specific cell types under specific conditions. Noncoding sequences are not included.

Question 23

Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot, one generally ________.

Answer 23

hybridizes filter-bound DNA with a DNA probe

Question 24

Northern blots are used to study what type of molecule?

Answer 24

RNA

RNA fragments are separated using gel electrophoresis and blotted onto a nylon membrane. Labeled nucleic acid fragments are used to probe the filter and identify hybridizing sequences.

Question 25

What is a probe in molecular biology?

Answer 25

A DNA or an RNA molecule used in hybridization reactions

A probe is a labeled single strand of DNA or RNA used to locate its complementary sequence. They are commonly used in southern and northern blots, as well as in library screening.

Question 26

Which of the following molecules is not required for a PCR reaction?

Answer 26

Ligase

Ligase is not required for a PCR reaction. The enzyme used during PCR is a thermostable DNA polymerase.

Question 27

TRUE or FALSE?

The thermostability of Taq polymerase is required during the annealing phase of PCR.

Answer 27

False

The annealing phase takes place at the lowest temperature of PCR. Taq polymerase is derived from bacteria that live in hot springs, so the enzyme is thermostable, meaning that its enzymatic properties can withstand the high temperatures needed for denaturation.

Question 28

What is the purpose of raising the temperature to 90–95°C at the beginning of each cycle of PCR?

Answer 28

To separate the double‑stranded DNA

The temperature is raised to denature the double‑stranded DNA molecule into single strands.

Question 29

The role of the primers in PCR is _______.

Answer 29

to define the target region and provide a 3’ end that can be extended by taq polymerase

Primers bind to end of the target DNA strands, then taq polymerase synthesizes a new strand using the target DNA as a template.

Question 30

If there are five molecules of DNA containing the target region at the beginning of a PCR reaction, how many copies of the target will be present after three rounds of amplification?

Answer 30

40

The number of target sequences is doubled with each replication cycle.

Question 31

Immediately after the primers have annealed to the target sequence, _______.

Answer 31

the temperature is raised so that taq polymerase can extend the primers

The temperature is raised to 70–75∘C, the temperature over which taq polymerase is optimally active.

Question 32

TRUE or FALSE?

During a PCR, heat is provided to inactivate the polymerase enzyme.

Answer 32

False

Question 33

In a typical PCR, primers are used to cleave specific regions of the DNA template.

Answer 33

False

Question 34

The products of restriction digestion can be visualized by gel electrophoresis, which separates fragments based on their size.

Answer 34

True

Restriction digestion produces fragments of DNA, and the sizes of these fragments can be determined by gel electrophoresis using standard DNA fragments of known size.

Question 35

A 1.5‑kb fragment of DNA is cloned into a plasmid vector that is 5.5 kb long at the EcoRI site, and the plasmid vector is then used to transform bacteria. If the plasmid DNA is then extracted from a single bacterial colony and digested with EcoRI, what digestion products will be produced if the plasmid contains the fragment?

Answer 35

One 1.5‑kb fragment and one 5.5‑kb fragment

EcoRI digestion will produce two fragments corresponding in size to the 1.5‑kb fragment cloned into the plasmid plus the 5.5‑kb plasmid itself.

Question 36

Digestion of a 1.1‑kb fragment of DNA with BamHI produces two fragments of 700 bp and 400 bp. Digestion of the same 1.1‑kb fragment with XhoI produces two fragments of 300 bp and 800 bp. Digestion with both enzymes produces three fragments of 100 bp, 300 bp, and 700 bp. Which of the following statements is true about the DNA fragment?

Answer 36

The XhoI site is located within the BamHI 400‑bp fragment.

Since double digestion produces a 100‑bp fragment and a 300‑bp fragment, the XhoI site must be located within the BamHI 400‑bp fragment.

Question 37

Which of the following statements about ddNTPs is true?

Answer 37

They have a hydrogen at the 3′ carbon of the sugar.

ddNTPs terminate synthesis because there is no 3′‑hydroxyl group onto which DNA polymerase can add nucleotides.

Question 38

DNA fragments that are 600 bp long will migrate more quickly through a sequencing gel than fragments that are 150 bp long.

Answer 38

False

Small DNA fragments have less hindrance in moving through the gel, so they migrate more quickly than larger fragments.

Question 39

Which of the following statements about manual Sanger sequencing is true?

Answer 39

The DNA sequence obtained is complementary to the template strand.

The DNA fragments produced in sequencing reactions are synthesized by DNA polymerase to be complementary to the template strand.

Question 40

A ddNTP, used often in DNA sequencing, lacks a(n) ________ at the ________ and ________ carbons.

Answer 40

OH; 2’; 3’

Question 41

The function of a ddNTP in DNA sequencing is to methylate guanine.

Answer 41

False

Question 42

In which of the following biochemical reactions is it common to use ddNTPs (dideoxyribonucleoside triphosphates)?

Answer 42

DNA sequencing

Question 43

Pyrosequencing is a method of DNA sequencing that uses beads as a matrix for PCR.

Answer 43

True

Question 44

The use of restriction enzymes
In recombinant DNA technology, restriction enzymes are used to isolate DNA segments (often genes) and recombine them with other pieces of DNA. A restriction enzyme recognizes a specific DNA base sequence—called a restriction site or recognition site—and cuts eachstrand of the DNA at a particular point within that site.

Answer 44

Most restriction sites are symmetrical: On each strand within the site, the sequence of bases is the same when read in the 5’ → 3’direction. The restriction site for the enzyme PvuI is an example of a symmetrical site.

Question 45

What are sticky ends?

Answer 45

A restriction enzyme cuts each strand of a DNA molecule at a particular point within the enzyme’s restriction site. If the cuts are staggered—that is, if the cut points on the two strands are not directly opposite each other—the resulting DNA fragments have single-stranded ends.These ends are called “sticky ends” because they can base-pair with complementary single-stranded ends on other DNA fragments.

Question 46

How can scientists produce a DNA fragment that contains a specific gene?

To clone a specific gene, scientists start with a much longer stretch of DNA containing the gene and use a restriction enzyme to cut theDNA into many fragments. Only one of these fragments—the one that contains the whole gene—is useful for cloning. This fragment must have two sticky ends to be integrated into a cloning vector, which happens in the next step.

To produce a DNA fragment that contains the whole gene and has two sticky ends, where must the enzyme’s restriction sites belocated?

Answer 46

outside the gene on both sides, but not inside the gene

Question 47

Restriction Enzymes, Recombinant DNA, and Gene Cloning
DNA cloning is a technique for producing many copies of a DNA segment for use in research, medicine, agriculture, or other applications.
In this tutorial, you will explore the procedures involved in cloning an imaginary gene in humans. DNA cloning is based on the following sequence of steps:

Answer 47

1. use of restriction enzymes to cut DNA into fragments
2. insertion and ligation of DNA fragments into a cloning vector
3. transformation and selection of recombinant bacteria
4. isolation of desired recombinant bacteria: screening the library

Question 48

Using restriction enzymes to cut DNA

The ability of restriction enzymes to cut DNA at specific sites makes DNA cloning possible.
The diagram below shows a section of human DNA that contains an imaginary gene for video game proficiency (the vgp gene), shown in red. Shaded areas mark the restriction sites (also called recognition sites) of four restriction enzymes: EcoRI, HaeIII, BamHI, and HindIII. Arrows indicate where each enzyme cuts the two DNA strands.

Which enzyme(s) will produce a DNA fragment that contains the entire vgp gene (shown in red) and has “sticky ends”?

Answer 48

BamHI, HindIII

Cloning the vgp gene requires an enzyme that has restriction sites on both sides of that gene but not within the gene. Two enzymes, HindIII and BamHI, satisfy that requirement, while also producing DNA fragments that have sticky ends. Sticky ends make it possible for the fragments to combine with the DNA of a cloning vector, such as a plasmid.

Question 49

BamHI cuts the plasmid outside the ampR gene, leaving the gene intact and functional, which is important for the next step in the cloning procedure. Once BamHI opens the plasmid, the sticky ends it produces in the plasmid can base-pair with those on BamHI human DNA fragments. If DNA ligase is then added, it will covalently join the sugar-phosphate backbones of the plasmid and the fragments, producing recombinant plasmids.

Answer 49

Note that human DNA contains many BamHI restriction sites in addition to the two shown in Part A. Therefore, only a small fraction of the recombinant plasmids will have the vgp gene, like the plasmid shown above. Most of the recombinant plasmids will have a fragment of human DNA that does not contain the vgp gene, and would not be of interest to the scientists.

Question 50

Which enzymes would cut the human DNA on both sides of the vgp gene, but not inside the gene?

Answer 50

BamHI , Haelll and HindIIl

Question 51

Which enzymes would cut the plasmid without disrupting the function of the amp^R gene?

Answer 51

BamHI, EcoRI, and HaeIII,

Question 52

Which enzymes would produce sticky ends when cutting both the human DNA and the plasmid?

Answer 52

BamHI, EcoRI, and HindIII,

Question 53

Which one restriction enzyme satisfies all three of the following requirements:

Would cut the human DNA on both sides of the vgp gene, but not inside the gene.

Would cut the plasmid without disrupting the function of the amp^R gene.

Would produce sticky ends when cutting both the human DNA and the plasmid.

Answer 53

BamHI

Question 54

What kind of plasmid will ONLY grow in medium without ampicillin:

Answer 54

no plasmid!

Question 55

What kind of plasmid will grow in both media?

Answer 55

nonerecombinant plasmid,

recombinant plasmid but no vgp gene,

recombinant plasmid with vgp gen

Question 56

Only bacteria that were transformed (picked up a plasmid) can grow in a medium containing ampicillin. Each transformed bacterium reproduces, forming a clone of cells.

Answer 56

The complete set of clones is called a plasmid library. Some clones in the library have nonrecombinant plasmids, some have recombinant plasmids without the vgp gene, and a small fraction have recombinant plasmids with the vgp gene.

Question 57

To screen a library of bacterial colonies for clones that carry a specific gene, a relatively short, single-stranded nucleic acid probe is hybridized to the DNA of that gene. Often, the probe is a DNA strand synthesized by reverse transcriptase from the mRNA encoded by the gene.

Answer 57

Bases in the probe hydrogen-bond to complementary bases in the gene. If the probe has been labeled with a radioactive isotope or a fluorescent tag, researchers can identify the bacterial clones that contain the DNA to which the probe has bound. Those clones can be grown in large quantities, allowing many copies of the gene to be isolated for use in research or other applications.

Question 58

Modern techniques

Answer 58

Developed in 1970’s

Virtual revolution in biotechnology

First commercial approval was 1982 for bacterial produced human insulin

Has led to an explosion of this industry

Question 59

In recombinant DNA technology, restriction enzymes are used to isolate DNA segments (often genes) and recombine them with other pieces of DNA. A restriction enzyme recognizes a specific DNA base sequence—called a restriction site or recognition site—and cuts each strand of the DNA at a particular point within that site.

Answer 59

Most restriction sites are symmetrical: On each strand within the site, the sequence of bases is the same when read in the 5’ → 3’ direction. The restriction site for the enzyme PvuI is an example of a symmetrical site.

Question 60

A restriction enzyme cuts each strand of a DNA molecule at a particular point within the enzyme’s restriction site. If the cuts are staggered—that is, if the cut points on the two strands are not directly opposite each other—the resulting DNA fragments have single-stranded ends.

Answer 60

These ends are called “sticky ends” because they can base-pair with complementary single-stranded ends on other DNA fragments.

Question 61

How can scientists produce a DNA fragment that contains a specific gene?

Answer 61

To clone a specific gene, scientists start with a much longer stretch of DNA containing the gene and use a restriction enzyme to cut the DNA into many fragments. Only one of these fragments—the one that contains the whole gene—is useful for cloning. This fragment must have two sticky ends to be integrated into a cloning vector, which happens in the next step.

Question 62

To produce a DNA fragment that contains the whole gene and has two sticky ends, where must the enzyme’s restriction sites be located?

Answer 62

outside the gene on both sides, but not inside the gene

Question 63

An enzyme will cut the human DNA on both sides of the vgp gene only if it has restriction sites to the left and right of the gene.

Answer 63

An enzyme will cut inside the vgp gene only if it has a restriction site within the gene.

Question 64

A gene in a bacterial plasmid consists of a continuous sequence of bases that is transcribed into mRNA, which is translated into a protein. Thus, the function of the gene is to code for a specific protein.

Answer 64

In the case of the ampR gene, the gene codes for a protein that enables bacteria to grow in media containing the antibiotic ampicillin.

Question 65

Suppose that a restriction enzyme cut the plasmid within the ampR gene and that an additional piece of DNA were inserted into the gene’s base sequence.

How would that affect the mRNA and ultimately the protein encoded by the ampR gene?

Answer 65

Both the mRNA and the protein would be abnormal.

Question 66

Sticky ends are single-stranded stretches at the ends of a DNA molecule that are formed when restriction enzymes make staggered cuts in DNA.

Answer 66

Sticky ends are single-stranded stretches at the ends of a DNA molecule that are formed when restriction enzymes make staggered cuts in DNA.

Question 67

Which of the following correctly describes a transformed bacterium?

Answer 67

a bacterium that has taken up external DNA, such as a plasmid

Question 68

How do ampicillin-sensitive bacteria become resistant to ampicillin?

The third step in the cloning procedure involves incubating ampicillin-sensitive bacteria with plasmids, some of which contain the vgp gene.

How do some of these bacteria become resistant to ampicillin?

Answer 68

by being transformed

Question 69

More than 200 restriction enzymes have been found in bacteria to date.

Answer 69

Many restriction endonucleases, including BamHI, recognize a palindromic sequence.

Question 70

Restriction enzymes cut DNA at specific recognition sites.

Answer 70

Many restriction sites are symmetrical about their midpoint.

Question 71

X-gal is a colorless substrate for the enzyme ____

Answer 71

X-gal is a colorless substrate for the enzyme beta-galactosidase

Question 72

A restriction enzyme that leaves either 5’ or 3’ overhangs produce “sticky ends.”

Answer 72

Restriction enzymes that leave no overhang produce blunt ends.

Question 73

Recombinant DNA is created by combining DNA molecules that are not found together naturally. This manipulation of DNA is achieved through the use of restriction endonucleases, which are enzymes that recognize specific palindromic DNA sequences.

Answer 73

Once both strands of the DNA within that sequence are cut by the restriction enzyme, the complementary tails of the DNA fragments can be annealed together by DNA ligase. More than 200 restriction enzymes have been identified in bacteria to date.

Question 74

Cloning involves the insertion of DNA fragments into vectors, which are agents that transfer genetic material from one cell to another.

Answer 74

Vectors are used to propagate and manipulate DNA fragments in bacteriophages and bacteria.

Question 75

Recombinant DNA is created by digesting DNA molecules with restriction enzymes and inserting them into carrier molecules called vectors.

Answer 75

Vectors can be derived from many sources, including bacterial plasmids and phages.

Question 76

Phage lambda (λ) is a bacteriophage whose central gene cluster can be replaced with a large foreign DNA fragment.

Answer 76

The phages are used to infect bacterial cells, where they replicate and form plaques from which the cloned DNA can be recovered.

Question 77

Smaller DNA fragments can be cloned into plasmids–naturally occurring extrachromosomal DNA molecules that replicate autonomously within bacterial cells. Up to 1000 copies of a plasmid may be produced within a single bacterial cell, so many copies of the cloned DNA can be generated.

Answer 77

Plasmid vectors generally contain a polylinker composed of unique restriction sites to facilitate the insertion of a foreign DNA fragment and genetic markers to allow for the easy detection of bacterial colonies that contain recombinant plasmids.

Question 78

The efficiency of transformation of bacterial plasmids starts to decrease at around 15 kb.

Answer 78

A piece of foreign DNA generally disrupts and inactivates the gene into which it is inserted.

Question 79

A clone library is a set of DNA segments derived from an individual.

Answer 79

It may represent an entire genome, a single chromosome, or a set of genes that are actively transcribed in a single cell type. A clone library can be generated and the techniques used to isolate specific clones from the library.

Question 80

cDNA libraries use mRNA as a starting point and thus represent only the expressed (transcribed) sequences under a given set of conditions. Because most eukaryotic mRNAs have poly-A tails at their 3’ ends, poly-dT oligonucleotides can be used as primers for the enzyme reverse transcriptase to synthesize cDNA strands complementary to the mRNAs. After degradation of the mRNA strands in mRNA/cDNA duplexes, DNA polymerase is used to synthesize complementary DNA strands. This results in double-stranded cDNA molecules that can be cloned into a suitable vector for the generation of the library.

Answer 80

Most methods for screening a cloned library involve hybridization with a DNA probe to identify specific clones, which then can be isolated and grown for further analysis. If the gene of interest has not been cloned but its approximate location on a chromosome is known, however, a technique known as chromosome walking can be used for several rounds of screening to recover overlapping clones from a genomic library until the gene in question has been reached.

Question 81

_______ involves the successive isolation of overlapping clones along a chromosome until the gene of interest is reached.

Answer 81

Chromosome walking involves the successive isolation of overlapping clones along a chromosome until the gene of interest is reached.

Question 82

Northern and Southern blots are used to identify_______.

Answer 82

Northern and Southern blots are used to identify nucleic acid sequences.

Question 83

Probes are used in several different molecular techniques, including southern and northern blots.

Answer 83

……

Question 84

The polymerase chain reaction (PCR) is a rapid, cell-free method of DNA cloning that has extended the power of recombinant DNA research.

Answer 84

The technique can exponentially amplify specific DNA sequences.

Question 85

PCR allows rapid direct amplification of specific target sequences of DNA that initially may be present in infinitesimally small quantities. The 3 steps in a PCR cycle are denaturation, annealing, and extension.

Answer 85

DNA molecules are first denatured by heating at 90-95ºC to dissociate the double-stranded molecules into single strands. During annealing, synthetic primers (oligonucleotides 15-30 nucleotides long) bind specifically to sequences flanking the segment to be amplified. These primers act as starting points for the synthesis of new DNA strands complementary to the target DNA. During extension, a heat-stable form of DNA polymerase, such as Taq polymerase, extends the primers by adding nucleotides in the 5’ to 3’ direction, making a double-stranded copy of the target DNA. Each PCR cycle theoretically doubles the number of copies of the target molecule, such that 25 cycles may result in more than a millionfold increase in the number of DNA copies.

Question 86

DNA sequencing is a method used to determine the nucleotide sequence of a DNA fragment.

Answer 86

This is the theory behind the most common method of sequencing and how it can be performed either manually or using an automated sequencer.

Question 87

The most commonly used DNA sequencing method, developed by James Sanger, involves the use of DNA polymerase to synthesize DNA strands repeatedly from a primer that binds close to the DNA to be sequenced. Fragments of different length are produced because strand synthesis terminates when a dideoxynucleotide (ddNTP) is incorporated into the growing strand. Sanger sequencing can be done manually or by using an automated sequencer.

Answer 87

In manual sequencing, 4 separate reactions are performed, 1 for each of the 4 ddNTP bases. These reactions are electrophoresed in separate lanes on a sequencing gel, which can then be read to reveal the base sequence of the DNA fragment.

An automated sequencer uses a single reaction mixture in which each of the 4 ddNTP terminating bases is labeled with a different fluorescent dye. The reaction is loaded in a single lane of a sequencing gel, and a laser reads the dye color terminating each fragment band and produces a graph from which the sequence is read directly.

Question 88

If the plasmid contains the fragment, EcoRI will cut in two places.

Answer 88

…..

Question 89

One of the first steps in characterizing a DNA clone is to produce a restriction map, which gives the position of restriction sites on a fragment of DNA.

Answer 89

Restriction mapping is a technique for determining the location of restriction sites on a fragment of DNA.

The DNA is digested with restriction enzymes, and the positions of the restriction sites are inferred from the sizes of the digested products.

DNA can be digested with restriction enzymes singly or in combination to determine the position of the restriction sites relative to one another.

The digested fragments can be resolved by gel electrophoresis, which separates fragments based on their size.

The fragment sizes (generally measured in thousands of base pairs, kb) are determined by comparison with DNA standards of known lengths.