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GENETICS LAB Final Review


Labs 1-3 Background
-Polymers: molecular arrays of simple repeating units
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-Nucleotides found in DNA and RNA are composed of a five carbon sugar, a nitrogenous
base and a phosphate group
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Rna
contains Uracil (U) instead of T
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The
backbone is sugar-phosphate
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-Alleles are different versions of the same gene
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-Three types of RNA: 1) mRNA- encodes a polypeptide and serves as a template for
translation
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3) rRNA- (ribosomal RNA) is both a structural and functional
component of ribosomes
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Coordinate
the process of translation and are basic functional units of the translational apparatus,
which also includes various soluble translation factors
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The mRNA translates and produces
polypeptides; amino acids
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-Chromosomes- highly ordered structures composed primarily of DNA and protein
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-Genome- array of chromosomal DNA inside of a cell
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-The haploid genome consists of one X or Y and 22 autosomes
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Results in genetically identical individuals called genetic clones
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-chromosomes are duplicated and sister chromatids are connected at the
centromere
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-Cytokinesis- division of the cytoplasm, is what yields two genetic clones
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-Reciprocal exchange of genetic material during meiosis increases genetic
variation
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Two
secondary meiotic cells are yielded
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The sister chromatids will
split
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Phenotype- is the measurable or
observable manifestation of genotype
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-Loss of function (LOF)- recessive allele does not encode functional product
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-Mendel’s Law of Segregation states that each individual diploid organism possesses two
alleles for any particular characteristic
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During
fertilization, two alleles randomly unite
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(adding fractions, don’t add the
denominators, just numerators
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(multiplying fractions,
multiply straight across, numerator and denominator
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-Genetic exchange can cause physically linked genes to appear unlinked in progeny
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2n, n=# of heterozygous gene pairs involved
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05

Lab 2
-Drosophila Melanogaster- useful for genetic research and has been used to define
fundamental genetic principles
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-The results of Morgan’s experiments led him to propose that genes are linked in a
linear array along chromosomes
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-Anabolic processes use energy to produce materials from simple substrates
-Catabolic processes involve degradation of complex compounds and release of energy
-Biochemical pathway- multi-step
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Series of chemical reactions occurring within a cell
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-Alkaptonuria- a rare metabolic disorder characterized by secretion of homogenistic acid
(HA) in urine
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This enzyme
normally catalyses the conversion of HA
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-Ommatidia are multiple visual structures on the eyes of the fruit fly
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Mutations in various genes can affect the
biochemical pathway leading to the red-brick eyecolor, resulting in different eye colors
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Melanogaster has 4 stages: egg, larva, pupae and adult
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-Larvae pass through three stages, or instars
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The adult fly
emerges as an imago, which is slender, long and pale
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-Almost entire genetic content of fruitfly resides on X,2,3 chromosomes
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-Flies are examined on stereo microscope (dissection microscope)
-Females are larger than males
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The males have darker bottoms than females
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Males have fewer sternites; bristled ventral abdominal
segments
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Lab 3
-Each cross began with true breeding (homozygous) parents
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Predicted ratios for F2 generations from a dihybrid cross (AaBb x
AaBb) is 9:3:3:1 phenotypic ratio and 1:1:2:2:4:2:2:1:1 genotypic ratio
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Separates DNA
fragments
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Ethidium bromide is an intercalating agent
that binds between bases
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Fragments of DNA
then appear as bands
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DNA migrates in a fashion
directly related to size
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Smaller fragments move more quickly, so larger molecules are closer to the top, and
smaller molecules are near the bottom
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This weighs down the sample and allows
for better visualization
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-Relaxed open circle of DNA may move faster or slower than linear DNA, depending on
concentration of agarose gel
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-RNA is usually on the bottom because it’s s smaller
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-The lanes that were treated with RNase look blank because the RNase broke down the
RNA
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-viruses and plasmids have the smallest genomes because they do not need the
genetic information required to encode an entire cell
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This will lyses the cells
efficiently
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Remove clear part
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-Remove protein by adding phenol/CHCl3
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-The phenol solution is saturated with a Tris buffer to ensure it does not absorb any
volume of the aqueous layer
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-principle is to utilize two oligonucleotide primers that hybridize, each to opposite
strands, at each end of the DNA region to be amplified
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-Temperature is lowered to ~50-62 C for primer to anneal to the complimentary sequence
in the DNA
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(Taq polymerase)
-One of these cycles doubles the amount of DNA
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-A specific heat insensitive form of DNA polymerase (Taq polymerase) from a hot spring
bacterium (Thermus aquaticus) is used for PCR so that new polymerase does not have to
be added at each cycle
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-Usually 3,000-5,000 base pair products can be efficiently amplified by Taq
polymerase
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-18S rRNA gene from the D
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-A PCR machine or thermal cycler is really just a programmable heat block that allows us to
control the temperature for each step that makes up a cycle of amplification
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Melanogaster) into a plasmid that can be grown in bacteria
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-Cloning is the first step for studying the structure and expression of genes
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-Cloning Steps: 1
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We did this by PCR amplification 2
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3
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Must have matching
“sticky ends” 4
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EX: the TOPO cloning plasmids have a
Topoisomerase 1 recognition site within rge MCS
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- Plasmids have an origin of replication that allows them to replicate in high copy
number within the bacterial cell
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- The energy is conserved by forming a phosphate bond with the cleaved strand and
tyrosyl residue (Tyr-274) of the topio
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- To harness he religating activity of topio, TOPO vectors provide linearized by topio
attached to a phosphate ate the 3’ end
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Coli
Transformation of Plasmid DNA into E
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- The cell is treated with CaCl2 (this speeds up the transformation process efficiency)
- Cells treated with CaCl2 are known as competent cells
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- The plasmid DNA is incubated at 42 C, this is known as the heat shock process
- Holes from in the membrane temporarily allowing plasmid DNA into the host cell
- The holes close and the cells replicate the new plasmid DNA
- These cells are placed on an ampicillin-containing agar medium (the cells containing
the plasmid should grow here b/c the plasmid is resistant to ampicillin)



Lab 8
Plasmid DNA minipreps
- Minipreps isolate plasmid DNA from small scale liquid cultures; sometimes the
colonies formed may not contain the plasmid DNA (false clones) but might be
resistant to ampicillin even though they do not contain the plasmid
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This step releases the DNA from the bacteria
- Add a third reagent potassium acetate (N3) which neutralizes the lysis reaction
- Centrifuge again
- Apply the solution to a column that binds specifically to DNA
- Wash the column to get rid of the proteins, salts, and buffers
- At the end, you end up with 50 microL of JUST plasmid DNA
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8 or greater is pure DNA or RNA
- How to find your concentration:
o (Your O
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X 50ng/micorL) / 1 O
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= your concentration
- Note: If your sample is diluted, you must multiply your sample by the volume you
added!
Restriction Enzyme Digestion of DNA
- Restriction enzymes: exonucleases that catalyze the cleavage of the phosphate
bonds w/in both strands of DNA
- Restriction enzymes only cut at recognition sites (specific base sequences)
- These enzymes protect bacteria from invading viruses
- They ONLY cleave dsDNA and leave “sticky ends”; ends that are complimentary to
each other; leaves complimentary overhangs
- EcoR1 and Hind3 are restriction enzymes used in this lab
Restriction Mapping
- Restriction mapping is a way to identify DNA fragments based on the principle that
any given DNA sequence has a unique pattern of restriction endonuclease sites
- We used this to make sure that the plasmid DNA isolated contained an insert of the
PCR gene fragment

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Each plasmid sample can be digested with appropriate restriction enzymes and then
visualized on an agarose gel

Lab 10
DNA sequencing
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DNA sequencing makes it possible to determine the order of nucleotides along a
fragment of DNA
The components are: 1
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Oligonucleotide primer 3
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Regular deoxyribonucleotides 5
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Buffer
containing Mg2+
Dideoxynucleotides are like deoxynucleotides except that do not have a 3’OH
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No further addition of nucleotides by Taq polymerase
2
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o The peaks on the left = shorter fragments
o The peaks on the right= longer fragments




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