MOLECULAR CLONING METHODS ● Stewart Linn and Werner Arber discovered restriction ●
endonucleases in E. coli in the late 1960s.
4.1 GENE CLONING ● These enzymes get their name from the fact that they prevent
A. THE ROLE OF RESTRICTION ENDONUCLEASES invasion by foreign DNA, such as viral DNA, by cutting it up. ●
● Thus, they “restrict” the host range of the virus.
B. VECTORS
● Furthermore, they cut at sites within the foreign DNA, rather than
C. IDENTIFYING A SPECIFIC CLONE WITH A SPECIFIC PROBE chewing it away at the ends, so we call them endonucleases ●
D. cDNA CLONING (Greek: endo, meaning within) rather than exonucleases (Greek:
exo, meaning outside).
E. RAPID AMPLIFICATION OF cDNA ENDS.
● Linn and Arber hoped that their enzymes would cut DNA at ●
4.2 THE POLYMERASE CHAIN REACTION specific sites, giving them finely honed molecular knives with which
to slice DNA.
A. STANDARD PCR
● Unfortunately, these particular enzymes did not fulfill that hope.
B. BOX 4.1 JURASSIK PARK: MORE FANTASY? ● However, an enzyme from Haemophilus influenzae strain Rd, ●
C. USING REVERSE TRANSCRIPTASE PCR (RT-PCR) discovered by Hamilton Smith, did show specificity in cutting DNA.
● This enzyme is called HindII (pronounced Hin-dee-two).
D. REAL-TIME PCR
● Restriction enzymes derive the first three letters of their names from
4.3 METHODS OF EXPRESSING CLONED GENES the Latin name of the microorganism that produces them.
● The first letter is the first letter of the genus and the next two letters
A. EXPRESSION VECTORS
are the first two letters of the species
B. OTHER EUKARYOTIC VECTORS ● (hence: Haemophilus influenzae yields Hin).
C. USING THE Ti PLASMID TO TRANSFER GENES TO PLANTS. ● In addition, the strain designation is sometimes included; in this
case, the “d” from Rd is used.
● Finally, if the strain of microorganism produces just one restriction
4.1 GENE CLONING
enzyme, the name ends with the Roman numeral I. If more than
● One product of any cloning experiment is a clone, a group of
one enzyme is produced, the others are numbered II, III, and so on
identical cells or organisms.
● HindII recognizes a specific DNA sequence:
● We know that some plants can be cloned simply by taking cuttings
○ GTPyPuAC
(Greek: klon, meaning twig), and that others can be cloned by
○ CAPuPyTG
growing whole plants from single cells collected from one plant.
● and cuts both DNA strands at the points shown by the arrows.
Even vertebrates can be cloned.
○ Py stands for either of the pyrimidines (T or C), and Pu
● John Gurdon produced clones of identical frogs by transplanting
stands for either purine (A or G).
nuclei from a single frog embryo to many enucleate eggs, and a
● Wherever this sequence occurs, and only when this sequence
sheep named Dolly was cloned in Scotland in 1997 using an
occurs,
enucleate egg and a nucleus from an adult sheep mammary gland.
● HindII will make a cut. Happily for molecular biologists,
● Identical twins constitute a natural clone.
● HindII turned out to be only one of hundreds of restriction
● The usual procedure in a gene cloning experiment is to place a
enzymes, each with its own specific recognition sequence. ●
foreign gene into bacterial cells, separate individual cells, and grow
● Table 4.1 lists the sources and recognition sequences for several
colonies from each of them.
popular restriction enzymes. ●
● All the cells in each colony are identical and will contain the foreign
, ● Note, for example, the complementarity between the ends created ● But what about DNA replication? Doesn’t that create newly ●
by EcoRI (pronounced Eeko R-1 or Echo R-1): replicated DNA strands that are unmethylated, and therefore
○ ↓ vulnerable to cleavage? Figure 4.1 explains how DNA continues to
59---GAATTC---39 ---G39 59AATTC— be protected during replication. ●
39---CTTAAG---59 ---CTTAA59 39G— ● Every time the cellular DNA replicates, one strand of the daughter
↑ duplex will be a newly made strand and will be unmethylated. ●
● Note also that EcoRI produces 4-base overhangs that protrude ● But the other will be a parental strand and therefore be methylated.
from the 59-ends of the fragments. This half-methylation (hemimethylation) is enough to protect the ●
● PstI cuts at the 39-ends of its recognition sequence, so it leaves DNA duplex against cleavage by the great majority of restriction
39-overhangs. endonucleases, so the methylase has time to find the site and
● SmaI cuts in the middle of its sequence, so it produces blunt ends methylate the other strand yielding fully methylated DNA. ●
with no overhangs.
● Restriction enzymes can make staggered cuts because the ●
sequences they recognize usually display twofold symmetry.
● That is, they are identical after rotating them 180 degrees. For
example, imagine inverting the EcoRI recognition sequence just
described:
○ ↓
59---GAATTC---39
39---CTTAAG---59
↑
● You can see it will still look the same after the inversion.
● In a way, these sequences read the same forward and backward.
● Thus, EcoRI cuts between the G and the A in the top strand (on
the left), and between the G and the A in the bottom strand (on the
right), as shown by the vertical arrows.
● Sequences with twofold symmetry are also called palindromes.
● In ordinary language, palindromes are sentences that read the
same forward and backward.
● Examples are Napoleon’s lament: “Able was I ere I saw Elba,” or a
Figure 4.1 Maintaining restriction endonuclease resistance after
wart remedy: “Straw? No, too stupid a fad; I put soot on warts,” or a DNA replication.
statement of preference in Italian food: “Go hang a salami! I’m a
lasagna hog.” ● We begin with an EcoRI site that is methylated (red) on both
● DNA palindromes also read the same forward and backward, but strands.
Fig
you have to be careful to read the same sense (59→39) in both ● After replication, the parental strand of each daughter DNA
directions. duplex remains methylated, but the newly made strand of each
● This means that you read the top strand left to right and the bottom duplex has not been methylated yet. ●
strand right to left. ● The one methylated strand in these hemimethylated DNAs is
● One final question about restriction enzymes: If they can cut up enough to protect both strands against cleavage by EcoRI. ●
endonucleases in E. coli in the late 1960s.
4.1 GENE CLONING ● These enzymes get their name from the fact that they prevent
A. THE ROLE OF RESTRICTION ENDONUCLEASES invasion by foreign DNA, such as viral DNA, by cutting it up. ●
● Thus, they “restrict” the host range of the virus.
B. VECTORS
● Furthermore, they cut at sites within the foreign DNA, rather than
C. IDENTIFYING A SPECIFIC CLONE WITH A SPECIFIC PROBE chewing it away at the ends, so we call them endonucleases ●
D. cDNA CLONING (Greek: endo, meaning within) rather than exonucleases (Greek:
exo, meaning outside).
E. RAPID AMPLIFICATION OF cDNA ENDS.
● Linn and Arber hoped that their enzymes would cut DNA at ●
4.2 THE POLYMERASE CHAIN REACTION specific sites, giving them finely honed molecular knives with which
to slice DNA.
A. STANDARD PCR
● Unfortunately, these particular enzymes did not fulfill that hope.
B. BOX 4.1 JURASSIK PARK: MORE FANTASY? ● However, an enzyme from Haemophilus influenzae strain Rd, ●
C. USING REVERSE TRANSCRIPTASE PCR (RT-PCR) discovered by Hamilton Smith, did show specificity in cutting DNA.
● This enzyme is called HindII (pronounced Hin-dee-two).
D. REAL-TIME PCR
● Restriction enzymes derive the first three letters of their names from
4.3 METHODS OF EXPRESSING CLONED GENES the Latin name of the microorganism that produces them.
● The first letter is the first letter of the genus and the next two letters
A. EXPRESSION VECTORS
are the first two letters of the species
B. OTHER EUKARYOTIC VECTORS ● (hence: Haemophilus influenzae yields Hin).
C. USING THE Ti PLASMID TO TRANSFER GENES TO PLANTS. ● In addition, the strain designation is sometimes included; in this
case, the “d” from Rd is used.
● Finally, if the strain of microorganism produces just one restriction
4.1 GENE CLONING
enzyme, the name ends with the Roman numeral I. If more than
● One product of any cloning experiment is a clone, a group of
one enzyme is produced, the others are numbered II, III, and so on
identical cells or organisms.
● HindII recognizes a specific DNA sequence:
● We know that some plants can be cloned simply by taking cuttings
○ GTPyPuAC
(Greek: klon, meaning twig), and that others can be cloned by
○ CAPuPyTG
growing whole plants from single cells collected from one plant.
● and cuts both DNA strands at the points shown by the arrows.
Even vertebrates can be cloned.
○ Py stands for either of the pyrimidines (T or C), and Pu
● John Gurdon produced clones of identical frogs by transplanting
stands for either purine (A or G).
nuclei from a single frog embryo to many enucleate eggs, and a
● Wherever this sequence occurs, and only when this sequence
sheep named Dolly was cloned in Scotland in 1997 using an
occurs,
enucleate egg and a nucleus from an adult sheep mammary gland.
● HindII will make a cut. Happily for molecular biologists,
● Identical twins constitute a natural clone.
● HindII turned out to be only one of hundreds of restriction
● The usual procedure in a gene cloning experiment is to place a
enzymes, each with its own specific recognition sequence. ●
foreign gene into bacterial cells, separate individual cells, and grow
● Table 4.1 lists the sources and recognition sequences for several
colonies from each of them.
popular restriction enzymes. ●
● All the cells in each colony are identical and will contain the foreign
, ● Note, for example, the complementarity between the ends created ● But what about DNA replication? Doesn’t that create newly ●
by EcoRI (pronounced Eeko R-1 or Echo R-1): replicated DNA strands that are unmethylated, and therefore
○ ↓ vulnerable to cleavage? Figure 4.1 explains how DNA continues to
59---GAATTC---39 ---G39 59AATTC— be protected during replication. ●
39---CTTAAG---59 ---CTTAA59 39G— ● Every time the cellular DNA replicates, one strand of the daughter
↑ duplex will be a newly made strand and will be unmethylated. ●
● Note also that EcoRI produces 4-base overhangs that protrude ● But the other will be a parental strand and therefore be methylated.
from the 59-ends of the fragments. This half-methylation (hemimethylation) is enough to protect the ●
● PstI cuts at the 39-ends of its recognition sequence, so it leaves DNA duplex against cleavage by the great majority of restriction
39-overhangs. endonucleases, so the methylase has time to find the site and
● SmaI cuts in the middle of its sequence, so it produces blunt ends methylate the other strand yielding fully methylated DNA. ●
with no overhangs.
● Restriction enzymes can make staggered cuts because the ●
sequences they recognize usually display twofold symmetry.
● That is, they are identical after rotating them 180 degrees. For
example, imagine inverting the EcoRI recognition sequence just
described:
○ ↓
59---GAATTC---39
39---CTTAAG---59
↑
● You can see it will still look the same after the inversion.
● In a way, these sequences read the same forward and backward.
● Thus, EcoRI cuts between the G and the A in the top strand (on
the left), and between the G and the A in the bottom strand (on the
right), as shown by the vertical arrows.
● Sequences with twofold symmetry are also called palindromes.
● In ordinary language, palindromes are sentences that read the
same forward and backward.
● Examples are Napoleon’s lament: “Able was I ere I saw Elba,” or a
Figure 4.1 Maintaining restriction endonuclease resistance after
wart remedy: “Straw? No, too stupid a fad; I put soot on warts,” or a DNA replication.
statement of preference in Italian food: “Go hang a salami! I’m a
lasagna hog.” ● We begin with an EcoRI site that is methylated (red) on both
● DNA palindromes also read the same forward and backward, but strands.
Fig
you have to be careful to read the same sense (59→39) in both ● After replication, the parental strand of each daughter DNA
directions. duplex remains methylated, but the newly made strand of each
● This means that you read the top strand left to right and the bottom duplex has not been methylated yet. ●
strand right to left. ● The one methylated strand in these hemimethylated DNAs is
● One final question about restriction enzymes: If they can cut up enough to protect both strands against cleavage by EcoRI. ●
