Module 1 Flashcards
(31 cards)
Natural Role of Restriction Endonucleases
Bacteria produce these enzymes to cleave (cut) double-stranded DNA at specific sequences to identify & eliminate foreign DNA, such as viruses.
Restriction Endonucleases NAMING
1st letter: Genus of bacterium
**Next 2 letters: ** species
**Number: **order the enzyme was discovered in each organism
Restriction Endonucleases Recognition
Recognize 4-6 base pair DNA sequences with rotational symmetry (palindromes)
Palindrome
One strand reads 5’ to 3’, other strand is its reverse complement.
Sticky Ends
- Staggered cuts, leaving single-stranded complementary sequences
- Form hydrogen bonds
Blunt Ends
- Cleave middle of sequence
- Do not form hydrogen bonds
Gel Electrophoresis
- Used to separate molecules like DNA and RNA by charge and size using agarose or polyacrylamide gel and an electric field
- Electric field is applied, molecules migrate through gel
- Smaller molecules move faster, larger molecules move slower
Steps in Gel Electrophoresis
1) Gel-electrophoresis apparatus
2) sieving action
3) visualization
Step 1 of gel electrophoresis
Gel-Electrophoresis Apparatus:
* Multiple samples are loaded into wells in a polyacrylamide gel
* Cover placed on gel → Voltage applied → negatively charged SDS (sodium dodecyl sulphate)-protein complex moves toward positive anode at bottom of gel
Step 2 of gel electrophoresis
Sieving Action
* The gel acts as a sieve, separating molecules by size
* Smaller molecules move fastest
Step 3 of gel electrophoresis
Visualization:
* After separation, DNA fragments formed from 4 different restriction enzyme digestions from 2 vial strains
* Stained with ethidium bromide → helps visualize the DNA bands
DNA Cloning
Method used to isolate DNA sequence -> introduced into cell to help amplify (increase production of) DNA sequence
DNA Cloning cleaves DNA using
specific endonuclease enzymes
What happens to DNA fragmens in DNA cloning
joined via DNA ligase to a cloning vector -> create hybrid molecule –> introduced into bacterial cell
Vector: transmits foreig DNA into cell
Applications of Cloned DNA
- Expressed to produce proteins
- Used for other DNA cloning experiments
- Isolated and analyzed
DNA Cloning Steps
- Digest 2 DNA sequences to produce same sticky ends
Ex. Plasmid Vector and PCR product of DNA - Mix the fragments; complementary sticky ends form hydrogen bonds
- DNA ligase forms phosphodiester bond between fragments
Plasmid
- Small circular DNA molecules
- Outside cell’s chromosomal DNA
- Restriction enzymes insert desired genes into plasmids & transfect them into mammalian cells
DNA Probes
- Single-stranded DNA molecules labelled with radioisotopes (e.g. phosphate 32,
32P) or non-radioactive labels (biotin or a fluorescent dye) - Help detect specific complementary DNA sequences (target DNA)
Hybridization (binding) of DNA Probe
DNA probe binds to target DNA → identifies sequence of interest among millions of other DNA sequences
Single-stranded oligonucleotide probes (20-30 nucleotides)
DNA Probe Hybridization
synthesized to be complementary to a specific region of the gene of interest
Amino acid sequence of a protein
DNA Probe Hybridization
used to design a probe by using genetic code as a guide
Allele-Specific Oligonucleotides (ASO) Function
for DNA Probes
Designed to bind to specific alleles (e.g., mutations or polymorphisms)
Allele-Specific Oligonucleotides (ASO) Application
For DNA Probes
Used in allele-specific PCR to detect if samples contain specific polymorphisms
DNA Probe Process
- DNA and ASO is mixed
- If the DNA contains the mutation, the probe binds and can be visualized
- If DNA of interest is low in concentration, PCR amplifys it, producing a detectable signal for mutation/polymorphism presence
