Genetics Midterm 2 Study Guide pt 2

Molecular Genetics
Field of biology that studies the structure and function of genes at a molecular level
Employs methods of genetics and molecular biology
Used to:
|| Understand genetic disorders
|| Rapidly detect genetic disoders
|| Criminology and Paternity testing
Extraction of Genomic DNA
DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis.
Three basic steps can vary:
|| Depending on the type of sample and interference of substances
|| Must be carried out in the presence of biological buffers if the end product is intended for latter use.
Three basic steps are:
|| Break open cells/remove membrane lipids through use of a detergent
|| Remove cellular/histone proteins bound to DNA through addition of a protease, precipitation with
sodium or ammonium acetate, or through use of a phenol/chloroform step.
|| Precipitate DNA in cold ethanol or IPA. DNA is insoluble in alcohol and clings together
+ This step also removes the salts added in step II.
Used solutions:
|| P1: 50 mM Tris-HCl, pH 8.0, 10 mM EDTA
|| P2: 20 mM NaOH, 1% Sodium Dodecyl Sulfate (SDS)
|| P3: 3.0 M potassium acetate
General Procedure:
|| Tris-HCl acts as buffer.
|| EDTA binds cations to weaken the cell envelope
|| SDS dissolves lipids and proteins
|| NaOH denatures DNA
|| Potassium acetate precipitates SDS, lipids and proteins
|| Precipitate is removed in a form of a pellet during centrifugation
|| Supernatant containing genomic DNA recovered from precipitation by IPA which also removes
remaining salts.
|| Final centrifugation leaves the pellets at the bottom of the Eppendorf tube.
When centrifuging, arrange the tube so that the hinge is up and the DNA pellet is underneath the hinge on the
side of the tube.


Genomic Extraction Protocol
250 uL P1 added to each Eppendorf tube with 8 flies
Flies ground.
250 uL P2, incubate at 65
o
C for 2 min
350 uL P3 solution, incubated on ice for 2 min
Centrifugation for 10 min
500 uL of clear lysate transferred.
800 uL of IPA (ice cold, 100%) added.
Centrifugation for 10 min
Place tube in heat block at 65
o
C until completely dry.
|| Too long and DNA will over dry, making it harder to dissolve.
150 uL of sterilized water was added and the tube mixed with the vortex mixer.
PCR
Creator: Kary Mullis
Commonly used:
|| Detection of hereditary diseases
|| Identification of genetic fingerprints
|| Diagnosis of infectious diseases
|| Cloning of Genes
|| Paternity Testing
|| DNA computing
Components of PCR:
|| Taq Polymerase: resists denaturation at high temperatures.
+ Denaturation of enzymes results in a loss of structure and function
|| dNTPs: 200 uM concentration
|| MgCl
2
: Taq polymerase requires the free magnesium ion to function. 1.5 mM concentration.
+ Forms a complex with dNTPs, which is then used as a substrate by polymerase
|| Template: target to be amplified
|| Primers: short ~20 bp single stranded pieces of designed sequence of DNA.
+ Specifically designed to bind to the two ends of the DNA segments being amplified.
|| Buffer: Solution containing Tris-HCl and KCl. Tris HCl maintains pH, 50 mM KCl provides optimum
conditions for polymerase and primer annealing.
Basic Steps (1 PCR cycle):
|| Denaturation: 94-96
o
C
|| Annealing: 48-56
o
C
|| Elongation: 64 72
o
C
Master mix contained all of the components except the template DNA.
Temperature Cycles:
1) 94
o
C for 2 min
2) 96
o
C for 10 sec
3) 57
o
C for 30 sec
4) 72
o
C for 2-4 min
5) repeat 2-4 30 times
6) 72
o
C 5 min
Purification of the PCR Product done by using QIAGEN QIAquick PCR purification Kit:
|| PE Solution: Ethanol
|| PB Solution (binding buffer): High Salt Concentration, Guanidine Hydrochloride, IPA
|| EB Solution (elution buffer): 10 mM Tris-Cl at pH 8.5
QUIAGEN Protocol:
PB -> PE (to wash bound DNA) -> EB
Quantification of DNA
Most accurate and quantitative method is using UV wavelengths obtained through a spectrophotometer
Quick, qualitative (inaccurate): compare unknown with known amounts of DNA.
|| Done by mixing both samples with ethidium bromide and measuring the intensity of fluorescence.
+ Fluorescence occurs because ethidium bromide intercalates between the bases of dsDNA.
DNA Gel Electrophoresis
Electrophoresis is used to separate macromolecules based on charge and size.
|| An electric field pushes negatively charged DNA molecules through a gel matrix.
Shorter DNA molecules move faster than longer ones, as they can slip through the matrix easier.
Lower the concentration of agarose, larger the ideal size of the sample molecule (aka inversely related)
Ranges from 50 bp to one million bp.
Visual comparison of the sample band intensity leads to an estimation of the amount of DNA loaded.
Uses negatively charged indicators:
|| bromophenol blue (runs @ 300 bp) (blue)
|| xylene cyanol (@4 kbp) (teal)
|| Orange G (@ 50 bp) (orange)
Glycerol, Ficoll (polysaccharide) or sucrose (used in the loading dye) increases density of the DNA sample to
sediment it. Ficoll was used in the loading buffer.
Electrophoresis: (-) -> (+)
Loading an Agarose Gel
Do not push the plunger past first stop (creates a bubble)
1 X Tris-acetate EDTA (TAE) electrophoresis run buffer poured into chamber; approximately 1-2 mm in height
All samples need loading buffer (6X concentration where 6X means 6 times diluted).
|| 5 uL of sample plus 1 uL of loading buffer
Order of well:
1 kb ladder, PCR unpurified (1, 2), PCR purified (1,2), + control, - control
Making agarose gel
400 mg electrophoresis grade agar
Mix with 40 mL 1X TAE and heat to a gentle boil. Swirl to make sure all is dissolved.
Pipette 4 uL of 10 g/L of ethidium bromide to the surface.
Place the gel combs on one end of the tray after pouring in the solution.
DNA and Protein Sequence Analysis
Sequence analysis is the study of DNA or peptide sequence using sequence databases.
It can:
|| Determine relatedness in form/function
|| Identify gene structures
|| Identify reading frames
|| Identify intron, exons and regulatory elements
Two processes of DNA sequencing:
|| Maxam/Gilbert Method:
+ chemical modification of DNA followed by subsequent cleavage.
|| Sangers Chain Termination Method:
+uses a fluorescent rather than radioactive tag.
Automated Sequencing v. PCR
Sequencing cant be done directly from genomic DNA, it requires multiple pure copies of it
Sequencing reaction uses only one primer; Each cycle produces only one strand of DNA
|| Replicated linearly instead of exponential in PCR
ddTTP = red, ddCTP = blue, ddGTP = yellow, ddATP = green
DNA Polymerase randomly incorporates ddNTPs or dNTPs.
End result is that fragments differ by only one nucleotide.
|| Polyacrylamide gels provide a dense matrix that can resolve single nucleotide differences
Automated sequencing results in a file of colored peaks (the chromatogram).
Base calling is when the computer assigns a nucleoside to each colored peak.
Problems:
|| Poor quality sequence (low peaks with high signal: noise ratio)
+results from impure DNA or low quantities of DNA or primer
|| Double peaks and scattered sections of noise
+result from mixed samples (heterozygote or multiple individuals)
OTHER
Methods to identify an unknown mutant allele:
Phenotypic analysis, Chromosomal/Allelic inheritance, gene location, DNA sequence analysis
Molecular approach identified the gen and mutation that gave rise to the visable phenotype
Theoretically, as small as a single DNA molecule could be used. Practically 10
3
10
4
used.
If dNTPs are not in equal molar concentration, mutations can occur.
QUIAGEN used a silica membrane column thats:
|| Easily subjected to quick centrifugation
|| High binding capacity
|| Easily fits most 1.7 mL microcentrifuge tubes.
Used 1.5% Agarose gel
Chaotropic molecules: those that disrupt/denature macromolecules
Buffer Types:
|| Running Buffer: Buffer in which the gel is made and used for electrophoresis
+ Tris-acetate-EDTA (TAE buffer)
|| Loading buffer: Added with DNA to monitor electrophoresis and sediment the sample
Characteristics of PCR Primers
About 18-30 bp long
Specific; complementary to just the section of DNA thats next to the region to be amplified
Have a melting temperature no more than 5
o
C difference between the pair (best between 1-2
o
C)
|| T
m
= 4(C + G) + 2(A + T) where G + C content should be 40-60%. Temperature in degrees.
There should be a GC Clamp: the 3 end of the primer should have one or two G or C bases in the last five bases.
Should have low intramolecular secondary structure formation; base pairing within itself.
Primer-Dimers: when primers base pair with itself.
No more than 4 repeats in a primer.
No more than a maximum number of single base runs of 4 (due to chance of misprime)

Orthologs: genes in different species evolving from the same ancestral gene
Paralogs: genes related by duplication within a genome (often evolve new functions).
--Drosophila melanogaster has homologs of the following mammalian proteins:
HSP70 chaperone, myosin, p53, and insulin
Protein motif (fingerprint): short, conserved region of a protein, usually 10-20 amino acid residues
Protein domain: region of a protein that can adopt a 3-D structure. Can be considered building blocks of proteins.
Protein motifs can be represented as a consensus or a profile. A zinc finger is a small protein structural motif that is
characterized by the coordination of one or more zinc ions in order to stabilize the fold.

Many types of Protein Domains:
Extend across the length of a protein
Occupy a subset of protein sequence
Occurs one or more times
Proteins that share only a domain might or might not be homologous:
Depends on function, mutant phenotypes, species similarity and other functions.
Missense mutations; still code for an amino acid, but the wrong one.
Nonsense mutations change an amino acid codon into a stop codon (leads to a non-functional protein almost always)