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Restriction Enzymes
1. What is a restriction enzyme?
This is an enzyme you can find in bacteria, this can cut the DNA of foreign
organisms and protects the cells from invaders such as viruses. These are also
known as restriction endonucleases.
2. What substrate does a restriction enzyme use?
The substrate or recognition sequence that a restriction enzyme uses are
symmetrical or palindromic, which means that the recognition sequence on one
DNA strand reads in the opposite direction on the complementary strand.
3. What is a recognition site?
The substrate for restriction enzymes are specific sequences of double stranded
DNA.
4. What is the difference between a blunt cut and a sticky cut?
A restriction enzyme recognizes and cuts DNA only at a particular sequence of
nucleotides. For example, the enzyme named HindIII incubated with EcoRI that
cuts DNA wherever it encounters the sequence, when the HaeIII restriction
enzyme cut straight across the double helix, it produces blunt cuts. Restriction
enzymes that cut in an offset fashion where the ends of the cut have an
overhanging piece of single-stranded DNA are sticky cuts.
5. How could you use a recognition site to cut out specific sections of DNA?
Using gel electrophoresis which is a procedure that separates molecules on the
basis of their rate of movement through a gel under the influence of an electrical
field. DNA is a negatively charged molecule, so it will move toward the positive
pole of the gel when a current is applied.
6. How are the sizes of the DNA fragments determined?
When DNA has been cut by restriction enzymes, the different-sized fragments
will migrate at different rates. Because the smallest fragments move the most
quickly, they will migrate the farthest during the time the current is on. The length
of each fragment will be measured by number of DNA pairs. If you know the
fragment sizes in the HindIII digest, you can use data from the marker to prepare
a standard curve, which will provide a standard for comparison to the unknown
fragment sizes.
7. In the analysis part of the lab the size of the fragments are measured. Graph this
information on the attached graph paper and determine the size of the fragments.
25000
20000
15000
10000
5000
0
10
13
15
20
29
32
47
8. What is the purpose of the standards? Why do we use semi-log paper to
determine the size of the fragments?
To stain DNA fragments for visualization and measure distances.
Do practice problems One and Two and write your answers:
#1
#2
Take the lab Quiz II and write your answers here:
1. The smaller a fragment, the farther it will migrate
b.
Migration distance is inversely proportional to the fragment size.
2. To calculate this, it is easiest to find the change in y at 10 minutes (0.4 ml - 0 ml =
0.4) and divided by the change in x (10 minutes - 0 minutes = 10 minutes). 0.4 ml/10
minutes = 0.04 ml/min.
b.
0.04 ml/min
3. If the restriction enzyme does not cut the DNA, a single large fragment will remain.
d.
The restriction enzyme EcoRI did not function properly
4.
c.
III
5. With EcoRI alone, there will be a single cut and only one fragment: 20kb
b.
II
6. When both BamHI and EcoRI are used, there will be four fragments: 2kb, 4kb, 6kb,
8kb.
e.
V
7.
a.
...GCGC
CGCG...
8. The fragments, in decreasing order of size are c, a, b. The DNA is negatively
charged, and loaded into wells at the negative electrode end.
b.
B
RFLPs
Define RFLP and how they are created?
RFLP stands for Restriction Fragment Length Polymorphism. Regions of the human
genome where restriction fragment lengths (RFL) are highly variable between
individuals, “P” stands for polymorphism, a Greek term that means "many shapes". The
lengths of some of the restriction fragments differ greatly between individuals, thus there
are many shapes, or lengths, of DNA possible.
Do both Blackett Activities and summarize each below. At the end of Activity One these
os a section called “Evaluating the DNA profiles”. Do questions 1-5 for each loci and
your results.
Go to activity Two and explain the difference between RFLPs and VNTRs.
VNTR stands for "variable number of tandem repeats". tandem repeat is a short
sequence of DNA that is repeated in a head-to-tail fashion at a specific chromosomal
locus. VNTRs are detected as RFLPs by Southern hybridization. If the DNA flanking a
VNTR is cut with a restriction endonuclease, the size of the resulting DNA fragment can
vary, resulting in RFLPs.
What is CODIS? Whose DNA is in CODIS. Could yours be?
Combined DNA Index System (CODIS) is the generic term used to describe the FBI ’s
program of support for criminal justice DNA databases as well as the software used to
run these databases. The National DNA Index System or NDIS is considered one part
of CODIS, the national level, containing the DNA profiles contributed by federal, state,
and local participating forensic laboratories. Mine is not there because it is a database
of DNA profiles, both from Criminal, or voluntary samples.
How are endonucleases used?
These restriction enzymes are found in many different strains of bacteria where their
biological role is to participate in cell defense.
What is an STR? What makes an STR suitable to be used in CODIS?
STR stands for Short Tandem Repeat, all CODIS STRs are tetrameric repeat
sequences. All forensic laboratories that use the CODIS system can contribute to a
national database. STR is suitable in CODIS because:

The CODIS system has been widely adopted by forensic DNA analysts
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STR alleles can be rapidly determined using commercially available kits.
STR alleles are discrete, and behave according to known principles of population
genetics
The data are digital, and therefore ideally suited for computer databases
Laboratories worldwide are contributing to the analysis of STR allele frequency in
different human populations
STR profiles can be determined with very small amounts of DNA
What does it means when on CSI they say the two people have 18 alleles in common?
A common use for a DNA test is to establish if a man is the biological father of a child, a
paternity test. If, for example, a child has two alleles that are designated 12.1 and 18,
and if the mother has alleles 12.1 and 16, then the child inherited the 12.1 allele from
the mother. The child has to have inherited the 18 allele from the father. The 18 allele is
the "obligate paternal allele." Generally, the alleged father must have this allele if he is
the biological father of the child.
How many loci need to be the same to determine wheter or not two people are parent
child? What about siblings?
When we see matching genotypes at all 13 CODIS loci, it is a virtual certainty that the
two DNA samples came from the same individual or an identical twin, between 9-11 loci
there is a strong parenthood relationship.
Do the attached activities and answer the questions.