DNA MAPPING using Restriction Enzymes Flashcards
Explain the mapping of DNA Mapping
- After restriction digests and mapping, the DNA Fragments can be sequenced
- Can be used to compare DNA sequences without having prior information about their nucleotide sequence
Knowing the location of restriction sites in a genome region of interest may be valuable for cloning parts of a gene into a vector or analyzing intron organization
Explain restriction enzymes
Restriction endonucleases are enzymes that recognizes a short and specific palindromic DNA sequence and cut (leave) it from inside
- they cleave both strands of DNA at sp3cific sequences
- the cleavage site is usually located within or near the recognition site
EcoRl cuts DNA at the sequence GAATTC
Describe the restriction enzyme nomenclature
HindIII H= Hemophilus(genus) in=influenzae(species ) d= d(strain) III= third endonuclease isolated from this bacteria
EcoRI E= escherichia (genus) co= coli(species) R= RY13(strain) I= first endonuclease isolated from this bacteria
Explain restriction enzyme mapping
- A restriction map is a map of known restriction sites within a sequence of DNA
- Done by breaking DNA into pieces and then identifying the locations of the break points
- The resulting fragments can be examined using gel electrophoresis, which is used to separate pieces of DNA according to their size
How can DNA be digested ?
In three ways
Two portions of the DNA sample are individually digested with different restriction enzymes
A third portion of the DNA sample is double-digested with both restriction enzymes at the same time
-the length of each individual DNA fragment depends upon the positions of its restriction sites, each restriction site can be mapped according to the lengths of the fragments
Explain each of the 2ays DNA can be digested
Single digest with enzyme ‘A’- enzyme ‘A’ will cut the DNA at its restriction site
In this example, the enzyme A has one restriction site on the plasmid. So…each plasmid is cut once, yielding linear DNA fragments
Single digest with enzyme ‘B’- will cut the DNA at its restriction site
In this example, the enzyme B has two restriction sites on the plasmid. So…each plasmid is cut twice, yielding DNA fragments of different sizes
Double digest with enzymes A and B-both will cut each plasmid DNA at their restriction sites
In this example, three fragments are obtained when both enzymes digest the DNA together
In double digest how can you use the pattern of fragments produced to find the relative locations of restriction sites?
Analyze the fragments produced by each single digest-The length of each individual DNA fragment depends upon the positions of its restriction sites
Option 1- match the length. Information from double digest is very useful in correctly mapping restriction sites
Option 2- this scenario also produces three fragments , but the sizes are different to those in option 1
The plasmid map drawn must yield the fragment pattern obtained during the experiment
What should be done after DNA digestion?
After digestion with DNA restriction endonuclease the DNA fragments can be separated according to size and shape by agarose or polysaccharide gel electrophoresis
-Gel electrophoresis can be used to determine the size of DNA molecules 500-30,000bp
Gel acts as a molecular sieve
DNA molecules have a net negative charge at neutral pH because of the phosphate group, so will migrate towards the positive electrode when an electric current is applied to DNA in a gel
Digested DNA fragments are run in parallel with a standard which contains fragments of known sizes
Explain the process of the restriction map
- Duplex DNA is detected by staining with intercalating substances like ethidium, acridium orange or profoavin, between stacked base pairs
- New stairs, like SYBR green, exhibit fluorescence under UV light
- As little as 50ng of DNA may be detected in a gel by staining it with ethidium bromide
- The final drawing of the DNA molecule showing the positions of the fragments is called the restriction map
What are the steps of restriction enzyme digestion?
- Label seven 1.5 ml microcentrifuge tubes with the numbers 2-8. Also label the tubes with your name or group identifier
- Add the reagents
- The enzymes EcoR1 and BamH1 will be the last reagents added to the tubes. You will get the enzymes from the lab technician
- After adding the reagents, mix solution by tapping or gentle vortexing
- Pulse spin (centrifuge) for 30-60 secs
- Incubate for a minimum of 2 hours in a 37 degrees Celsius water bath
- Set up gel tank- attach rubber damps to two sides of gel bed
- Attach well-forming comb
- Add a small amount of cooled agarose (55 degrees Celsius) to both inside ends of bed
- Wait for about 1 min fir agarose to solidify - Pouring cooled (55 C) agarose solution into tank, and allow to cool for 20 mins
- Removing rubber dams and comb
- Carefully remove rubber dams from both ends, one at a time
- Remove comb slowly and carefully, by pulling it straight up to avoid tearing the wells/gels
What should be done after incubation?
After 2 incubation, remove sample from water bath
- Add 5 uL of concentrated (10X ) gel loading solution to each tube. The sample can be stored in the freezer fir electrophoresis at a later time
- Cap and mix tubes by gentle invertion
- Heat the samples, including the Std DNA at 65 degrees Celsius for 2 minutes. Allow the samples to cool for a few minutes
- Load 20 uL of standard DNA fragment (std) into the first well
- Load all of the contents of each reaction sample (in consecutive order) into wells 2-4
How to cover electrophoresis tank?
Carefully snap down the cover into the electrode terminals
-Note that during electrophoresis, the DNA samples migrate through the agarose gel towards the positive electrode. Make sure the gel is properly oriented
