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They are type II restriction enzymes with a ommon structural core comprising four β-sheets and a single α-helix.
restriction endonucleases
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Which enzyme would cut the human DNA shown in Part A on both sides of the vgp gene but not inside the gene?
1. Which enzyme(s) would cut the human DNA shown in Part A on both sides of the vgp gene, but not inside the gene? Answer: BamHI, HaeIII, and HindIII 2. Which enzymes(s) would cut the plasmid without disrupting the function of the amp^R gene? Answer: BamHI, EcoRI, and HaeIII 3. Which enzyme(s) would produce sticky ends when cutting both the human DNA and the plasmid? Answer: BamHI, EcoRI, and HindIII 4. Which one restriction enzyme satisfies all three of the requirements listed above? Answer: BamHI only
WHAT IS pBR322?
pBR322 is one of the most used cloning vectors in molecular biology. Cloning vectors, best-known as plasmids, are autonomously replicating DNA units into which DNA fragments can be inserted for gene cloning. Genes taken up by these plasmids are multiplied (or cloned) as the vector replicates, to yields numbers suitable for molecular analysis. The most versatile and well-known plasmid is certainly pBR322 (in fact was one of the first ever used in gene cloning techniques) and has genetically tailored cutting sites into which DNA can be inserted without affecting plasmid self-replication. pBR322 general characteristics are: a) Size: 4.3 kb; b) Replicon: ColE1, relaxed; c) Selective markers (resistance): Amp and Tet; d) Single sites (enzymatic restriction single sites): Ava I, Pst I, BamHI, PvuII, ClaI, SalI, EcoRI, and HindIII.
Function of hindiii?
Haemophilus influenzae is a species of bacteria that is the source of the HindIII restriction enzyme that cleaves the palindromic DNA sequence 5'-AAGCTT-3' in the presence of the cofactor Mg2+ via hydrolysis. While restriction enzymes cleave at specific DNA sequences, they are first required to bind non-specifically with the DNA backbone before localizing to the restriction site. On average, the restriction enzyme will form 15-20 hydrogen bonds with the bases of the recognition sequence. With the aid of other Van der Waals interactions, this bonding facilitates a conformational change of the DNA-enzyme complex which leads to the activation of catalytic centers. Despite the lack of evidence suggesting an exact mechanism for the cleavage of DNA by HindIII, site-mutagenesis analysis coupled with more detailed studies of metal ion-mediated catalysis in EcoRV have led to the following proposed catalytic mechanism. It has been suggested that during the hydrolysis of DNA by EcoRV the catalytic residue Lys-92 stabilizes and orients the attacking water nucleophile, while the carboxylate of Asp-90 stabilizes the leaving hydroxide anion through to coordination of Mg2
What is the difference between Puc18 and Puc19?
The multiple cloning sites (MCS) in pUC18 and pUC19 is the difference - the MCS in pUC19 is reverse orientated to those of the pUC18.pUC18: LacZ HindIII PaeI ..... SacI EcoRIpUC19: Lacz EcoRI SacI ..... PaeI HindIII
What is a degradative enzyme that recognizes specific nucleotide sequences and cuts up DNA?
Those are restriction enzymes, a kind of endonucleases. Some wellknown ones are EcoRI, HindIII or BamHI
What group of organisms provides a source of restriction enzymes for genetic engineering?
Bacteria. The enzymes are typically named after the bacteria also. For example EcoRI comes from E. coli and HindIII comes from H. influenzae.
What is the role of ECORI?
It splices the genome or plasmid in a specific location (EcoRI).
Which enzyme would cut the human DNA shown in Part A on both sides of the vgp gene but not inside the gene?
1. Which enzyme(s) would cut the human DNA shown in Part A on both sides of the vgp gene, but not inside the gene? Answer: BamHI, HaeIII, and HindIII 2. Which enzymes(s) would cut the plasmid without disrupting the function of the amp^R gene? Answer: BamHI, EcoRI, and HaeIII 3. Which enzyme(s) would produce sticky ends when cutting both the human DNA and the plasmid? Answer: BamHI, EcoRI, and HindIII 4. Which one restriction enzyme satisfies all three of the requirements listed above? Answer: BamHI only
What is the role of EcoRI enzyme?
It splices the genome or plasmid in a specific location (EcoRI).
What is Example of restriction enzyme?
A restriction enzyme (also known as restriction endonuclease) is protein which cuts DNA up at specific sequences (called restriction sites) in a genome. For example, the commonly used restriction endonuclease EcoRI recognizes every DNA sequence GAATTC and cuts at the point between the guanine and the adenine in that sequence, forming blunt ends (or straight, even ends). Interestingly and coincidentially, the restriction site for most restriction enzymes are genetic palindromes (the sequence reads exactly the same backwards on the complementary strand). In the case of EcoRI, the two complementary DNA strands for the restriction site are:5'-- GAATTC --3'3'-- CTTAAG --5'After this DNA sequence is cut, it might look something like this:5'-- G AATTC --3'3'-- C TTAAG --5'
If you took a linear piece of DNA and cut it with the restriction enzyme EcoRI and it had three restriction sites for EcoRI, how many fragments would you produce What if you had a circular piece of DNA?
If the plasmid has 3 recognition sequences for a given restriction endonuclease, then 4 linear DNA fragments are obtained because, if the DNA is linear then the number of fragments obtained is (N+1) whereas if the DNA is circular then the number of fragments obtained will be N for N recognition sequences for the given restriction endonuclease in a plasmid.
WHAT IS pBR322?
pBR322 is one of the most used cloning vectors in molecular biology. Cloning vectors, best-known as plasmids, are autonomously replicating DNA units into which DNA fragments can be inserted for gene cloning. Genes taken up by these plasmids are multiplied (or cloned) as the vector replicates, to yields numbers suitable for molecular analysis. The most versatile and well-known plasmid is certainly pBR322 (in fact was one of the first ever used in gene cloning techniques) and has genetically tailored cutting sites into which DNA can be inserted without affecting plasmid self-replication. pBR322 general characteristics are: a) Size: 4.3 kb; b) Replicon: ColE1, relaxed; c) Selective markers (resistance): Amp and Tet; d) Single sites (enzymatic restriction single sites): Ava I, Pst I, BamHI, PvuII, ClaI, SalI, EcoRI, and HindIII.
Function of hindiii?
Haemophilus influenzae is a species of bacteria that is the source of the HindIII restriction enzyme that cleaves the palindromic DNA sequence 5'-AAGCTT-3' in the presence of the cofactor Mg2+ via hydrolysis. While restriction enzymes cleave at specific DNA sequences, they are first required to bind non-specifically with the DNA backbone before localizing to the restriction site. On average, the restriction enzyme will form 15-20 hydrogen bonds with the bases of the recognition sequence. With the aid of other Van der Waals interactions, this bonding facilitates a conformational change of the DNA-enzyme complex which leads to the activation of catalytic centers. Despite the lack of evidence suggesting an exact mechanism for the cleavage of DNA by HindIII, site-mutagenesis analysis coupled with more detailed studies of metal ion-mediated catalysis in EcoRV have led to the following proposed catalytic mechanism. It has been suggested that during the hydrolysis of DNA by EcoRV the catalytic residue Lys-92 stabilizes and orients the attacking water nucleophile, while the carboxylate of Asp-90 stabilizes the leaving hydroxide anion through to coordination of Mg2
The plasmid sample is supposed to contain only the DNA fragmentWhy are there more than one band?
Probably the plasmid is partially uncutted and it is present in his coiled and supercoiled forms. you can add another control as the undigested construct. In addition you can add much more enzyme, change batch of EcoRI and digest overnight with BSA to exclude star activity.
Why is it important to inactivate the shrimp alkaline phosphatase before ligating your EcoRI cut pBluescript plasmid to a foreign DNA fragment?
because you need the phosphate of the foreign DNA fragment for ligation. If not inactivated before you add the fragment, SAP will imediately remove the phosphate form your fregment too leaving you with two dephosphorylated fragments that are not easy to ligate..
