BIO 121 Final

Helicase

An enzyme that UNZIPS the double helix at the replication forks, separating the two parental strands and making them available as template strands.

Primers are

Short, single-stranded sequence of RNA or DNA that enables the start of replication of a DNA sequence that is synthesized from the 3' end of the primer. Two types are needed, a forward and a reverse primer.

A type of heat stable DNA-polymerase used in PCR

Taq

Blasting

The National Institute of Health (NIH) and National Center for Biotechnology Information (NCBI) hosts a database called GenBank, which houses all known DNA sequences. Once the sequences of our samples are ready, they are pasted into a search tool (called a BLAST) which matches them to the correct species

What is the last step of PCR?

extension

Pearson's r is used to illustrate the relationship between

two continuous variables, such as years of education completed and income. The correlation between any two variables using Pearson's r will always be between -1 and +1. A correlation coefficient of 0 means that there is no relationship, either positive or negative, between these two variables. A correlation coefficient of +1 means that there is a perfect positive correlation, or relationship, between these two variables.

Animal barcoding studies

use a region in the mitochondrial cytochrome c oxidase 1 gene ("CO1") or the cytochrome b gene (CYTb).

Plant barcoding studies

use one or a few plastid regions and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA.

Photosynthesis reactants

water & carbon dioxide; 12 H2O + 6 O2

Which of the following is true about our positive control for DNA Analysis?

- If it's incorrectly identified all our results are invalid and we would need to start all over again with fresh extractions. - It should always show up as expected in your gel (at the right length). - If it's incorrectly identified all our results are invalid and we would need to start all over again with fresh extractions.

If your P value is less than the chosen significance level then you reject the null hypothesis

- i.e. accept that your sample gives reasonable evidence to support the alternative hypothesis. It does NOT imply a "meaningful" or "important" difference; that is for you to decide when considering the real-world relevance of your result.

Steps in DNA Analysis

1. Sample processing, 2. Digestion & extraction, 3.PCR, 4. Gel electrophoresis, 5. Sequencing, 6. Blasting

scientific method

1. ask a question, 2. conduct bckgrnd research, 3. form hypothesis, 4. test, 5. analyze data/draw conclusions, 6. communicate results

Photosynthesis formula

6CO2 + 6H2O + sunlight ---> C6 H12 O6 + 6O2

Taq polymerases are

A type of heat-stable DNA polymerase derived from a species of bacteria living in hot springs. Because Taq polymerase continues to function normally at high temperatures, using it allows researchers to separate the DNA strands without destroying the polymerase.

gel electrophoresis

Agarose gel electrophoresis is a method used to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length. Using electricity, the DNA (with a - charge) is pushed through the gel towards the positive electrode. As your gel "runs," the DNA is separated by size. The DNA strands show up as bands under UV light and you can read the results. Your products can be compared with the ladder or marker, which has standard sized DNA fragments of KNOWN length used for comparison. In this way, you can know the exact length of your DNA samples

A cytochrome is

An iron-containing protein that is a component of electron transport chains in the mitochondria and chloroplasts of eukaryotic cells and the plasma membranes of prokaryotic cells

Sample Processing

Bushmeat Processing: Using aseptic techniques, the bushmeat samples are cut into approximately 1 cc sections. They labeled and stored in ethanol at -20 degrees Celsius. Special care is taken to ensure no cross-contamination or human contamination occurs. Samples are then carried back or shipped to WKU

A complete list of the products of cellular respiration:

CO2, H2O and energy.

A metabolic process by which cells secure energy from various compounds is

Cellular respiration.

Which process is the most efficient?

Cellular respiration.

Cytochrome b

Cytochrome c is the most stable and abundant member, where it has been studied the most. Found in mtDNA, cytochrome b plays an imperative role in the mitochondria and kicks off complex III in the electron transport chain. These cytochromes help convert food into a form that cells can use. They are found in eukaryotic cells in the mitochondrion.

Digestion & Extraction

Digestion liquefies the tissue in such a way that keeps the DNA intact for extraction. We use special "kits" as pictured to streamline the process. Extraction requires more steps to break through the cell and organelle membranes to free the DNA. Once extraction is complete, the DNA sample is tested to ensure an adequate amount of intact DNA was extracted from the sample.

T or F. Anabolic reactions breakdown food to obtain energy.

FALSE

T or F. Catabolic reactions synthesize large molecules from smaller ones.

FALSE

A complete list of reactants in cellular respiration:

Glucose and oxygen.

p value significance

Most authors refer to statistically significant as P < 0.05 and statistically highly significant as P < 0.001 (less than one in a thousand chance of being wrong).

Mix buffer

Necessary to create optimal conditions for activity of Taq DNA polymerase and may contain restriction enzymes, which act like molecular scissors cutting the copied DNA strands at particular locations based on their genetic code.

Sequencing

Once we know we have amplified (copied) the right gene we are ready to sequence the gene. We expect the sequence (the order of As, Ts, Cs and Gs) within the cytochrome b gene to be different for different species. Samples are placed into a sequencer apparatus which can detect the order of nucleotide bases in our sample. The sequence is then cleaned and edited

polymerase chain reaction (PCR)

PCR makes copies of a DNA fragment from one original copy. The goal is to amplify a specific region, the target DNA or gene of interest (GoI), depending on the type or goal of research. The PCR cocktail includes the following ingredients: the DNA sample, primers (short sequences of RNA or DNA that start replication), dNTPs (free nucleotides), taq polymerase (a heat stable form of DNA polymerase derived from bacteria) and a buffer solution. There are three steps to PCR in which the temperature is cycled. The total # of resulting DNA strands is (the number of original strands) X 2^n, where n = the number of PCR cycles.

Villages samples are from

Rukanga, Jora, Bungule, Mwakasinyi, Keteghe

​Agarose gel electrophoresis is

a method used in molecular biology to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length.

1. How many different things are placed in the PCR tube? List each PCR component and what is does.

a. DNA Sample - provides the genetic information to be coded from sequence b. Primers - single stranded sequences of RNA or DNA as a forward and reverse to form hydrogen bonds at complementary ends of the target DNA. c. DNA Polymerase - "Taq" is heat-stable in high temperatures and is allowing research for DNA separation without destroying the polymerase d. Nucleotides - Freely in place to be used to construct new DNA copies e. Mix buffer - molecular scissors cutting the DNA copy at specific locations in genetic code f. PCR Tube - proper equipment used in the DNA extraction and holding for sequence to code and take place

What type of gel will we/would we have create(d) in lab?

agarose

What is the second step of PCR?

annealing

We will use a search tool called a ____________________ to match our samples in a big DNA database called ____________________ which houses all known DNA sequences.

blast; genebank

You can study the rate of cellular respiration by measuring

both the products and reactants.

We are attempting to get the DNA sequence of our gene of interest (GoI), which is _____________________.

cytochrome b

What is the first step of PCR?

denaturation

This semester's samples were sourced from 8 butcheries-

from villages of Rukanga, Jora, and Bungule

Snared species from bushmeat

gazelle, dik dik, zebra, & impalas

Which of the following is NOT contain in a PCR solution?

gel buffer

Photosynthesis products

glucose & oxygen; 6 H2O + 6 O2 + C6H12O6

The negatively charged phosphate groups of the sugar-phosphate backbone of DNA will migrate

in an electric field away from the negative side (top) and toward the positive electrode (bottom).

DNA polymerase

is the enzyme primarily responsible for carrying out the process of DNA synthesis. There are several different types of DNA polymerase which have different functions throughout.

Pearson's correlation coefficient (r)

is used to demonstrate whether two variables are correlated or related to each other.

A pathway used by animal cells in oxygen deficient conditions is

lactate fermentation.

Standard sized DNA fragments used for comparison in gels are called

ladders

dNTPs are

monomers that serve as DNA building blocks.

Shorter DNA molecules

move faster and migrate further down the gel.

What type of charge does DNA have?

negative

What is the pH indicator we will use in this experiment?

phenolphthalein

What does PCR stand for

polymerase chain reaction

To which electrode will DNA migrate in gel electrophoresis?

positive

To get the code from our DNA samples, they are placed into a _____________ which can detect the order of nucleotide bases in our sample.

sequencer

Gel electrophoresis separates DNA fragments by what?

size

Longer ones migrate

slower remain closer to the top of the gel.

What is our titrant in this experiment?

sulfuric acid

What is cycled through the various steps of PCR

temperature