Baylor University Genetics Lab Test 2 Study Guide

What is the name of Lab 6?

Gel Electrophoresis, Ligation, and Transformation

Agarose gel electrophoresis

Separates DNA fragments based on their size

What is loading dye used for?

1) provides color to visually track sample

2) makes sample heavy so it sinks into wells

When an electrical current is run through a gel, which direction do the DNA fragments travel?

The DNA fragments travel towards the positive pole at the opposite end of the gel from the wells.

Why aspect of DNA makes it move through the gel?

The negatively charged phosphate backbone causes the DNA fragments to move towards the positive pole.

What size of DNA fragments will be closer to the positive pole?

The small fragments will be closer to the positive pole, because they are lighter and move through the gel faster than heavier fragments.

What type of an agent is a DNA stain?

An intercalating agent

What does an intercalating agent do?

It wedges its between the bases of the DNA double helix

How can you make a DNA stain fluoresce?

You can view it under ultraviolet light

What DNA stain did we use in this lab?

GelRed

What is GelRed known as?

A mutagen or intercalating agent

What is a cloning vector?

carries the DNA into the E. Coli.

What is a plasmid?

Small, circular pieces of DNA that are extrachromosomal (replicated independently of the chromosome). The plasmid is the cloning vector in the lab.

Multiple Cloning Site (MCS)?

contains the plasmid's restriction enzymes

Selectable markers

Aid in identifying bacterial cells that have taken up a plasmid and which of those that are recombinant (carrying a foreign DNA insert).

What are the selectable markers in the pUC cloning vector?

Ampr and lacZ

How do you create a recombinant plasmid?

The foreign DNA and the plasmid must be cut with the same restriction enzyme so they yield sticky ends

Ligation reaction

The enzyme ligase seals the pieces of DNA to the plasmid through the creation of phosphodiester bonds.

What is transformation?

The process in which the E. coli takes up the plasmid

What method do we use to transform the bacterial cells?

Heat shock

How does the heat shock method allow transformation to progress?

It creates pores in the cell's membrane which also the ligated plasmid to enter the E. coli cell.

What is the purpose of the Ampr selectable marker in the plasmid?

Checks if the E. coli has taken up a plasmid. The ampr maker makes the bacteria resistant to the antibiotic ampicillin.

What is the purpose of the lacZ selectable marker in the plasmid?

Checks if the the transformed plasmid has a DNA insert. The lacZ gene is disrupted when the DNA insert is ligated. Therefore, beta-gal cannot be created to break down the X-gal that is on the plate. This results in the bacterial colonies maintaining their white color

What is the title of Lab 7?

Screening Transformed Colonies and PCR

What is the purpose of a Polymerase Chain Reaction (PCR)?

A PCR amplifies millions of copies of DNA from a very small starting amount.

What is denaturation?

The first step of a PCR that heats the DNA to 95 degrees C. to break the hydrogen bonds been base pairs.

What is annealing?

The second step of a PCR that lowers the temp to 30-65 degrees C. so that primers (3'-OH) can bind to the two DNA template strands.

What are the names of the two primers needed during annealing?

Forward and Reverse primers

What is elongation/extension?

The third step of a PCR that raises the temp to 72 degrees C. so that dNTP's can be added (complementary and antiparallel) to the growing DNA chain.

What DNA polymerase is used in a PCR elongation step?

Taq polymerase

Why is taq polymerase important?

It is isolated from a thermal bacterium and therefore and can stay stable in the high temperature environments that the DNA is subject to in denaturation.

How many times are the three PCR steps repeated?

Usually around 30 times

What machine revolutionized PCR efficiency?

thermocyclers

What other two ingredients are needed in a PCR?

1) MgCl2 - activated Taq

2) Buffer solution

What is transformation efficiency (TE)?

TE is a measure of ow well a ligation/transformation reaction worked at taking up a plasmid

What is a master mix?

A mixture of all the necessary reagents that go into a PCR minus the DNA.

What is a benefit of using a master mix?

You can pipet large volumes, which creates greater precision and accuracy. It also allows for greater consistency between samples.

How much extra master mix do you want to make?

10% more than the reaction calls for

What ingredients make up the 1% ;agarose gel?

0.5g agarose + 50mL buffer + 1XTAE

What is the title of lab 8?

Gel Electrophoresis, purification, and sequencing of PCR products

What method are we using to purify the DNA from our PCR reaction?

Exonuclease-phosphatase (Exo-AP) method

What does phosphatase do in the purification process?

Inactivates any remaining dNTPs from the PCR by cataloging the removal of the 5' phosphate group of the dNTP

What does the exonuclease do in the purification process?

Degrades any remaining primers by cataloging the removal of nucleotides from primers (3'-5' direction)

What is the Sanger method?

Amplifies the DNA fragment much like a PCR reaction

What makes the Sanger method different than a PCR?

There are both dNTPs (used in PCR) and ddNTPs ( not used in PCR) in the solution. A ddNTP terminates elongation of the DNA chain and thus chains of different lengths are created by the random incorporation of ddNTPs.

How can we detect when a ddNTP is present?

The 4 types of ddNTPs each have a different color fluorescent dye that can be read by a laser scanner.

What makes the Sanger method preferable over other next-gen sequencing technologies?

Sanger method is highly accurate over long lengths of DNA. Between 500-1000 nucleotides

What ingredients did the company add to our DNA samples during their sequencing of our purified PCR product?

(DNA and primer) + dNTPs, ddNTPs, DNA polymerase, and buffer

What is the purpose of a DNA mass ladder?

It allows you to estimate the size of fragment and concentration of PCR product

What is the title of Lab 9?

Bioinformatics

What does BLAST stand for?

Basic Local Alignment Search Tool

What is the goal of bioinformatics?

To organize, analyze, and store sequence data

What does it mean to annotate a gene sequence?

The RNA and amino acid sequences the gene encodes are determined.

What is comparative genomics?

Sequence function and location of a gene are evaluated across different species.

What does homologous mean in terms of how two genes are related?

That two genes are likely evolutionarily related to one another and most likely have a similar function.

What is the title of Lab 10?

An Introduction to Population Genetics

What is population genetics?

Division of genetics that explores the genetic composition of populations and how that changes over time.

What is a Mendelian population?

A population that consists of sexually reproducing individuals that are able to interbreed.

What is a gene pool?

The set of genes within the population

What is a genotypic frequency?

The proportion of a particular genotype within a population

How do you calculate genotypic frequency?

(# of individuals with certain phenotype) / (total # of individuals in the population)

What is an allelic frequency?

The proportion of a particular allele within a population.

How do you calculate allelic frequency?

(# of copies of allele in population) / 2( total # of individuals in population)

What does Hardy-Weinberg Law state?

If certain conditions are met, allelic frequencies in a population will not change from generation to generation.

Hardy-Weinberg Equilibrium

What are the conditions for Hardy-Weinberg to apply?

1) The population is infinitely large

2) the members of the population mate randomly

3) mutations do not occur

4) migration does not occur

5) natural selection does not occur

What is genetic drift?

Happens when allelic frequencies change with time due to a population being to small

What are three mechanisms that can cause a sample size to become small and therefore cause genetic drifts to occur?

1) limited resources

2) founder effect

3) bottleneck effect

What is a founder effect?

A small number of individuals from one population leave and establish a new population.

What is a bottleneck effect?

A population becomes reduced in number

What type of markers did we amplify when we swabbed our cheek cells?

Alu transposable elements (PV92 and TPA25)

What is the title of Lab 11?

Studying Evolutionary Genetics with Mitochondrial DNA

What are some unique properties of mitochondrial DNA?

1) It is a circular molecule

2) thousands of copies of mtDNA in a cell

3) mtDNA less prone to degradation

4) Uniparental (usually maternal) inheritance

5) Rapid mutation rate

What is a haplogroup?

The specific version of co-inherited genetic variation an individual carries.

What causes mtDNA to mutate more frequently than other types of DNA?

1) Fewer repair mechanisms

2) Proximity to reactive oxygen species which are mutagenic

Why are mtDNA sequences between individuals not drastically different despite the high rate of mutation?

The highly variable regions of mtDNA are located in a non-coding control region.

What does mtDNA primarily code for?

ATP (cellular energy), tRNA and rRNA (protein synthesis)

How does natural selection limit the accumulation of changes over time in mtDNA?

It purges deleterious mutations that disrupt mtDNA's vital functions.

What is a fixed allele?

An allele that has been homogenized for an entire population.

What is sequence divergence?

A measure of how many bases or amino acids differ between two lineages.

What is a molecular clock?

The tracking of how mutations in mtDNA accumulate at a relatively constant rate. This allows us to date past evolutionary events.

What is the Out-of-Africa origin theory?

The theory that the common ancestor of all modern humans arose out of East Africa

What evidence is there for the Out-of-Africa theory?

1) more variation in mtDNA found in Africa than the rest of the world combined

2) variations found outside Africa are subsets of those found in Africa

3) All modern humans more closely related to each other than to other species

What is the purpose of a phylogenetic tree?

To depict the inferred relationships between taxa