Lab 10 Flashcards
DNA
(Deoxyribonucleic Acid)
3 sources:
- bacterial genomic DNA
- bacterial plasmid DNA
- eukaryotic DNA
Chemical structure is the same
= two long strands of nucleotides (adenine (A), cytosine (C), guanine (G) and thymine (T)) that are connected by the sugar-phosphate backbone.
= double helix due H-bonds between complementary nucleotides (A-T and G-C).
–> if H-bond between them broken, 2 strands of helix come apart = denatured DNA
Number of base pairs will refer to the size of different pieces of DNA.
For example, 3000 base pairs (bp) or 3 kilobase pairs (kbp or simply kb) implies the piece of DNA has 3000 nucleotides on each strand in the double helix.
DNA in prokaryotic cells
Genomic (also referred to as chromosomal) DNA in E.coli (a species of bacteria) consists of a double stranded DNA helix arranged in a circle that is anchored to the bacterial plasma membrane.
- approximately 3,000 - 4,000 genes located on this DNA molecule.
- genes on this DNA encode all the functions a bacterial cell needs to survive.
Bacteria often have plasmid DNA in addition to Genomic DNA
- Bacterial plasmid DNA is also a circular molecule, but it floats freely in the cytoplasm of the bacterial cell.
- contain between 2 and 25 genes.
- these genes are not required for the survival of the cell but confer a selective advantage to cells under certain conditions.
= In the lab the plasmid we will extract carries a gene that allows the bacteria to grow in the presence of an antibiotic (for example: ampicillin or kanamycin).
- Plasmids are much smaller than genomic DNA
- and are supercoiled (tightly coiled), a feature that is key to enabling their extraction from the other cellular components.
DNA in eukaryotic cells
Eukaryotic DNA is arranged in linear strands called chromosomes that are located in the nucleus of the cell.
- Humans have 23 pairs of chromosomes or 46 chromosomes in total.
–> Human DNA contains approximately 21,000 different genes.
mini prep
This procedure allows one to isolate plasmid DNA from bacterial cultures quickly and at low cost.
- procedure discriminates between plasmid DNA and bacterial genomic DNA based on the size (plasmids are smaller than the genomic DNA).
- and conformation of the DNA molecules (plasmids are supercoiled and floating in the cytoplasm, whereas the genomic DNA is not supercoiled and is anchored to the plasma membrane).
Cautions with mini prep procedure
- If the bacterial genomic DNA is broken into small pieces, it will not be removed by precipitation with the rest of the genomic DNA.
THEREFORE, MUST MIX TUBE VERY GENTLY. - RNA also a very small nucleic acid molecule, gets removed by RNAse during the mini-prep procedure (RNAse is in the P1 solution).
Enzyme composition
- Specifically folded into 3-dimensional shapes – including the critical active site shape for substrate binding and reaction progression.
- Shape is held together by many types of bonds that cumulatively are effective but individually are very susceptible to the pH and temperature environment.
Restriction enzymes
- are proteins that recognize specific sequences of DNA and cleave the DNA within that sequence.
- contain active sites to bind DNA at specific DNA sequences and cut the phosphodiester backbone.
- produced naturally by some bacteria.
- Different strains of bacteria produce different restriction enzymes.
- can use restriction enzymes to cut and paste DNA from one source into another.
restriction enzyme, HindIII
will use HindIII to cut the plasmid DNA you extracted.
- HindIII recognizes 6 nucleotide bases: 5 ’– AAGCTT –3’.
Everywhere this specific combination of bases occurs in the DNA , HindIII cuts the phosphodiester backbone of the DNA strands between the first two adenines
Restriction digests are a diagnostic tool that allows the differentiation between plasmid based on the size of fragments generated from the digest.
Precise conditions for restriction enzymes
To cut DNA with a restriction enzyme, simply add the enzyme to the DNA and incubate the reaction for an hour.
Digestion, however, must take place under very specific conditions of salt concentration, pH and temperature.
- conditions depend upon restriction enzyme.
- ensure conditions met = concentrated buffer specific for the enzyme is added to the DNA-restriction enzyme reaction.
The fragments of DNA
Restriction enzymes will cut a piece of DNA into discrete fragments of varying size, depending on the location and number of the recognition sites.
- fragments appear as bands when they are separated by gel electrophoresis.
–> and the size of the fragments can be determined by the migration distances of the bands on the gel.
A restriction map
is a diagram showing the location of the restriction cut sites and the distances between them in a piece of DNA .
- in lab, will run DNA gels to determine the size of fragments generated by the restriction digest of your plasmid DNA .
–> will allow you to identify the mystery plasmid. - Once determine the sizes of the fragments in your digests, can confirm identity of Plasmid using restriction maps.
Antibiotics
are molecules that kill or inhibit the growth of microorganisms such as bacteria.
Plasmids often contain a gene(s) that make the bacterial cell resistant to a specific antibiotic.
Ampicillin
antibiotic commonly used to treat bacterial infections in humans.
Works as an antibiotic antibiotic by preventing the synthesis of cell walls in some bacteria.
- AmpR gene = DNA sequence the the bacteria transcribes into mRNA and translates into a protein.
- the protein functions as an enzyme, called beta-lactamase, that inactivates ampicillin [antibiotic], thereby protecting the bacteria from cell death.
