Bio Lecture 22 Flashcards
What are restriction enzymes or restriction endonucleases?
Restriction enzymes are bacterial enzymes that cleave dsDNA in a specific way. Cuts at specific sequences and patterns. Can leave sticky ends or blunt ends depending on how it cuts. Usually involves palindromes where it cuts.
What is a vector?
Circular molecule of dsDNA that is capable of autonomous replication in a host cell.
Has three minimum requirements:
-Antibiotic resistance to select for the cells with the new DNA
-Origin of replication to allow replication of the vector
-Multiple cloning site with one or more palindromes
How is DNA cloned using bacteria?
Take a vector, use a restriction enzyme to cut it open.
Take the DNA you want to replicate with compatible sticky ends to the cut vector.
Insert new DNA into the vector and let the bacteria replicate all it wants.
What is Gel Electrophoresis?
Way of separating nucleic acids based on their mass.
Sample placed in a gel and an electric charge is applied.
DNA/RNA are neg charged and therefore move towards the pos pole.
Smallest particles move the fastest.
What are the two gels used for gel electrophoresis?
Agarose: larger pore size, separates molecules that are several hundred base pairs different
Polyacrylamide: smaller pore size, separates down to one base pair difference
How does the Sanger DNA sequencing method work?
dideoxynucleotides are used instead of normal dNTPs
These lack a 3’ OH group so the replication stops after this nucleotide is inserted.
A template strand is added to the mixture as well as dNTPs and a few ddNTPs and a labeled primer and DNA-polymerase.
A different double deoxy base is run in each lane so the replication will only stop after those bases in those lanes. Run the mixtures in four lanes in polyacrylamide gel. Read the results from the bottom to the top to get the sequence of the DNA.
How does automated sequencing work?
The same as the Sanger method, but instead of using a labeled primer, each ddNTP type is given a different fluorescent dye that a computer can pick up on and record. This means that only one lane is needed to do the sequencing.
What are the benefits of PCR?
Fast, specific to any region of DNA you want, sensitive down to very small samples
What is PCR and how does it work?
Polymerase Chain Reaction
It is a way to copy double stranded DNA.
In one tube add: template strand, 2 sets of primers, many dNTPs, heat-stable DNA-polymerase (Taq polymerase).
Use a thermocycle machine to run many heating and cooling cycles to denature and renature the strands of DNA.
Each cycle doubles the original amount of DNA. So the total produced is 2^n after n-cycles.
Steps are: denaturation, annealing, elongation
What is RT-PCR?
Allows production of DNA from mRNA.
First step is to use reverse transcriptase to make an intron only copy of the DNA. Then, PCR is run like normal.
What is blotting and what are the types?
Blotting is a way to detect specific sequences or proteins in a mixture.
Southern: for DNA
Northern: for RNA
Western: for Protein
Samples are separated with gel electrophoresis, then placed on a paper and the test is run. If present, a band will appear.
What are microarrays?
Machines with silica slides that contain many different genes. It tests for the presence of genes so you can know what form of a protein is being produced or what allele is present.
