restriction mapping - purdue university
TRANSCRIPT
RESTRICTION MAPPINGRESTRICTION MAPPING
AN INTRODUCTORY NOTE How to show the nucleotide sequence of a piece of DNA
Example: 5’-ATGGCTATG-3’…antisense strand: serves as template for RNA synthesis 3’-TACCGATAC-5’…sense strand: has the same sequence as the mRNA
To write down the nucleotide sequence, start from the 5’ end of the sense strand, as shown below:
5’-CATAGCCAT-3’ 3’-GTATCGGTA-5’
RESTRICTION MAPPING
Objectives: a) to cut the DNA into pieces b) to construct a restriction map for the DNA of
interest
RESTRICTION MAPPING
Objectives: a) to cut the DNA into pieces b) to construct a restriction map for the DNA of
interest
What is restriction map? Restriction map is the physical map of a DNA which shows the relative positions of restriction enzyme cleavage sites.
Gene number in experimental organisms
Drosophila melanogaster (fruit fly): 13,920 Homo sapiens (human): 22,287 Arabidopsis thaliana (thale cress): 27,655 Mus musculus (house mouse): 24,500 Phage lambda: 60 (= 48,502 base pairs)
Strategies to study the DNA: a) cut the DNA into pieces b) study the function of each piece c) re-assemble the pieces
In order to cut (=digest, cleave) the DNA, use restriction enzymes (= restriction endonucleases).
Restriction enzymes are extracted from bacteria: Type I, not a useful tool; see later Type II, widely used to cut the DNA
Species of bacteria make restriction enzymes which recognize palindromic (=inverted repeats ) nucleotide sequences of DNA, called restriction sites, and cleave the DNA at those sites generating a 5’ phosphate and a 3’ sugar group at the point of cleavage.
O Base 5’
O
Base
5’
O
P
O
5’
O
Base
O
OH
P
O
5’
OH
O
Base
1’
2’
3’
4’
Example 1. EcoRI (“eeko-R-one”), the first enzyme extracted from E. coli strain RY13. EcoRI recognizes the sequence GAATTC as shown below:
5’…TGCGCTAATTGAATTCGTAACTGATC…3’ 3’…ACGCGATTAACTTAAGCATTGACTAG…5’
5’…TGCGCTAATTG AATTCGTAACTGATC…3’ 3’…ACGCGATTAACTTAA GCATTGACTAG…5’
Example 2. HindIII (“hin-D-three”), the third enzyme extracted from Haemophilus influenza strain D. HindIII recognizes the sequence AAGCTT as shown below:
5’…TGCGCTAATTAAGCTTGTAACTGATC…3’ 3’…ACGCGATTAATTCGAACATTGACTAG…5’
5’…TGCGCTAATTA AGCTTGTAACTGATC…3’ 3’…ACGCGATTAATTCGA ACATTGACTAG…5’
Example 3. Occasionally when internal bases of two different restriction sites are identical, hybridization of nucleotides will take place, but the newly-generated site won’t be palindromic.
BamHI Bacillus amyloliquefaciens
Sau3AI Staphylococcus aureus
5’ GATC 3’ 3’ CTAG 5’
5’…TGCGCTAATTG GATCCGTAACTGATC…3’
3’…ACGCGATTAACCTAG GCATTGACTAG…5’
5’…ACGGCTATCT GATCTAAGCATCGT…3’
3’…TGCCGATAGACTAG ATTCGTAGCA…5’
Other examples:
PstI Providencia stuartii 5’ CTGCA G 3’ 3’ G ACGTC 5’
(staggered ends)
AluI Anthrobacter luteus 5’ AG CT 3’ 3’ TC GA 5’
(blunt ends)
These are Type II restriction enzymes.
Type I restriction enzymes recognize a specific sequence in the double-stranded DNA, but cut the DNA at a non-specific site away from the sequence. These enzymes are not useful for the analysis of DNA sequences.
Test yourself
Size: 1200 bp
310 400
The linear map of phage λ (lambda) DNA
0 10,000 20,000 30,000 40,000 48,502 bp A
0 21,226 26,104 31,747 39,168 44,972 48,502 bpEcoRI B
Fragment 21,226 4,873 5,643 7,421 5,804 3,530 size
HindIII C
25,157 37,459
Fragment 23,130 2,027 2,322 9,416 564 125 6,557 4,361 size
The linear map of phage λ (lambda) DNA
0 10,000 20,000 30,000 40,000 48,502 bp A
0 21,226 26,104 31,747 39,168 44,972 48,502 bpEcoRI B
Fragment 21,226 4,873 5,643 7,421 5,804 3,530 size
HindIII C
25,157 37,459
Fragment 23,130 2,027 2,322 9,416 564 125 6,557 4,361 size
21,226 26,104 31,747 39,168 44,972
23,130 27,479 36,895 37,584 44,141
25,157 37,459
EcoRI HindIII
1,904 947 4,268 564 1,584 831
The linear map of phage λ (lambda) DNA
0 10,000 20,000 30,000 40,000 48,502 bp A
0 21,226 26,104 31,747 39,168 44,972 48,502 bpEcoRI B
Fragment 21,226 4,873 5,643 7,421 5,804 3,530 size
HindIII C
25,157 37,459
Fragment 23,130 2,027 2,322 9,416 564 125 6,557 4,361 size
21,226 26,104 31,747 39,168 44,972
23,130 27,479 36,895 37,584 44,141
25,157 37,459
1,904 947 4,268 564 1,584 831
EcoRI HindIII 21226 23130
2027
21226 48502 5148/4973 4268 3530 2027 1904 1584 1373
947 831
1. Prepare digestion reactions as described in the Lab Manual.
E H EH NO
2. Incubate at 37°C for 45 min.
3. Add 5 μL of gel loading dye to each tube; mix well and centrifuge briefly.
EcoRI
DNA
Buffer
HindIII
DNA
Buffer
4. Load on gel as shown on right:
5. Electrophorese for 40 min at 90 volts.
6. Photograph the gel and identify the bands; the results must be consistent with the figure shown on right.
DNA ladder E H EH NO E H EH
How to Construct a Restriction Map
You have a piece of linear DNA of unknown size. You digest the DNA once with enzyme A, once with enzyme B, and once with enzymes A and B. Draw the restriction map of the DNA.
DNA Enzyme Enzyme Enzymes ladder A B A+B
7
3
8
7
3
8
7
3
8
7
3
8
7
3
8
7
3
8
Restriction map
0 10
RESTRICTION MAPPING
RESTRICTION MAPPING
RESTRICTION MAPPING
Slide Number 21
Slide Number 22
Slide Number 23
Slide Number 24
Slide Number 25
AN INTRODUCTORY NOTE How to show the nucleotide sequence of a piece of DNA
Example: 5’-ATGGCTATG-3’…antisense strand: serves as template for RNA synthesis 3’-TACCGATAC-5’…sense strand: has the same sequence as the mRNA
To write down the nucleotide sequence, start from the 5’ end of the sense strand, as shown below:
5’-CATAGCCAT-3’ 3’-GTATCGGTA-5’
RESTRICTION MAPPING
Objectives: a) to cut the DNA into pieces b) to construct a restriction map for the DNA of
interest
RESTRICTION MAPPING
Objectives: a) to cut the DNA into pieces b) to construct a restriction map for the DNA of
interest
What is restriction map? Restriction map is the physical map of a DNA which shows the relative positions of restriction enzyme cleavage sites.
Gene number in experimental organisms
Drosophila melanogaster (fruit fly): 13,920 Homo sapiens (human): 22,287 Arabidopsis thaliana (thale cress): 27,655 Mus musculus (house mouse): 24,500 Phage lambda: 60 (= 48,502 base pairs)
Strategies to study the DNA: a) cut the DNA into pieces b) study the function of each piece c) re-assemble the pieces
In order to cut (=digest, cleave) the DNA, use restriction enzymes (= restriction endonucleases).
Restriction enzymes are extracted from bacteria: Type I, not a useful tool; see later Type II, widely used to cut the DNA
Species of bacteria make restriction enzymes which recognize palindromic (=inverted repeats ) nucleotide sequences of DNA, called restriction sites, and cleave the DNA at those sites generating a 5’ phosphate and a 3’ sugar group at the point of cleavage.
O Base 5’
O
Base
5’
O
P
O
5’
O
Base
O
OH
P
O
5’
OH
O
Base
1’
2’
3’
4’
Example 1. EcoRI (“eeko-R-one”), the first enzyme extracted from E. coli strain RY13. EcoRI recognizes the sequence GAATTC as shown below:
5’…TGCGCTAATTGAATTCGTAACTGATC…3’ 3’…ACGCGATTAACTTAAGCATTGACTAG…5’
5’…TGCGCTAATTG AATTCGTAACTGATC…3’ 3’…ACGCGATTAACTTAA GCATTGACTAG…5’
Example 2. HindIII (“hin-D-three”), the third enzyme extracted from Haemophilus influenza strain D. HindIII recognizes the sequence AAGCTT as shown below:
5’…TGCGCTAATTAAGCTTGTAACTGATC…3’ 3’…ACGCGATTAATTCGAACATTGACTAG…5’
5’…TGCGCTAATTA AGCTTGTAACTGATC…3’ 3’…ACGCGATTAATTCGA ACATTGACTAG…5’
Example 3. Occasionally when internal bases of two different restriction sites are identical, hybridization of nucleotides will take place, but the newly-generated site won’t be palindromic.
BamHI Bacillus amyloliquefaciens
Sau3AI Staphylococcus aureus
5’ GATC 3’ 3’ CTAG 5’
5’…TGCGCTAATTG GATCCGTAACTGATC…3’
3’…ACGCGATTAACCTAG GCATTGACTAG…5’
5’…ACGGCTATCT GATCTAAGCATCGT…3’
3’…TGCCGATAGACTAG ATTCGTAGCA…5’
Other examples:
PstI Providencia stuartii 5’ CTGCA G 3’ 3’ G ACGTC 5’
(staggered ends)
AluI Anthrobacter luteus 5’ AG CT 3’ 3’ TC GA 5’
(blunt ends)
These are Type II restriction enzymes.
Type I restriction enzymes recognize a specific sequence in the double-stranded DNA, but cut the DNA at a non-specific site away from the sequence. These enzymes are not useful for the analysis of DNA sequences.
Test yourself
Size: 1200 bp
310 400
The linear map of phage λ (lambda) DNA
0 10,000 20,000 30,000 40,000 48,502 bp A
0 21,226 26,104 31,747 39,168 44,972 48,502 bpEcoRI B
Fragment 21,226 4,873 5,643 7,421 5,804 3,530 size
HindIII C
25,157 37,459
Fragment 23,130 2,027 2,322 9,416 564 125 6,557 4,361 size
The linear map of phage λ (lambda) DNA
0 10,000 20,000 30,000 40,000 48,502 bp A
0 21,226 26,104 31,747 39,168 44,972 48,502 bpEcoRI B
Fragment 21,226 4,873 5,643 7,421 5,804 3,530 size
HindIII C
25,157 37,459
Fragment 23,130 2,027 2,322 9,416 564 125 6,557 4,361 size
21,226 26,104 31,747 39,168 44,972
23,130 27,479 36,895 37,584 44,141
25,157 37,459
EcoRI HindIII
1,904 947 4,268 564 1,584 831
The linear map of phage λ (lambda) DNA
0 10,000 20,000 30,000 40,000 48,502 bp A
0 21,226 26,104 31,747 39,168 44,972 48,502 bpEcoRI B
Fragment 21,226 4,873 5,643 7,421 5,804 3,530 size
HindIII C
25,157 37,459
Fragment 23,130 2,027 2,322 9,416 564 125 6,557 4,361 size
21,226 26,104 31,747 39,168 44,972
23,130 27,479 36,895 37,584 44,141
25,157 37,459
1,904 947 4,268 564 1,584 831
EcoRI HindIII 21226 23130
2027
21226 48502 5148/4973 4268 3530 2027 1904 1584 1373
947 831
1. Prepare digestion reactions as described in the Lab Manual.
E H EH NO
2. Incubate at 37°C for 45 min.
3. Add 5 μL of gel loading dye to each tube; mix well and centrifuge briefly.
EcoRI
DNA
Buffer
HindIII
DNA
Buffer
4. Load on gel as shown on right:
5. Electrophorese for 40 min at 90 volts.
6. Photograph the gel and identify the bands; the results must be consistent with the figure shown on right.
DNA ladder E H EH NO E H EH
How to Construct a Restriction Map
You have a piece of linear DNA of unknown size. You digest the DNA once with enzyme A, once with enzyme B, and once with enzymes A and B. Draw the restriction map of the DNA.
DNA Enzyme Enzyme Enzymes ladder A B A+B
7
3
8
7
3
8
7
3
8
7
3
8
7
3
8
7
3
8
Restriction map
0 10
RESTRICTION MAPPING
RESTRICTION MAPPING
RESTRICTION MAPPING
Slide Number 21
Slide Number 22
Slide Number 23
Slide Number 24
Slide Number 25