TAMU bio 111 lab exam 2 review

What is the difference between the coding and non-coding region?

Coding region: comprised of genes that encode proteins (instructions that determine traits) and makes up 1-2% of human DNA. • Noncoding region: makes up 98-99% of human DNA, contains STRs and is responsible for regulatory functions such as gene transcription.

What are STRs?

Short-tandem repeats (STRs) are unique repeating patterns of the same nucleotide sequence. They can be used to differentiate one person from another.

What is a DNA profile?

A DNA profile is a specific pattern of DNA attributes that is obtained in the lab and can be used to identify an individual. • DNA profiling is the process of determining an individual's DNA characteristics (profile) from a sample of bodily tissue

What does PCR stand for? What is the purpose of PCR?

Polymerase Chain Reaction • Purpose is to amplify DNA. In forensics, this can be used to obtain more DNA from a sample that would otherwise be too small to analyze.

What is inside the master mix?

3 pairs of primers, loading dye, nucleotides (dNTPs), Taq polymerase, mix buffer, and water. DNA sample is added to master mix.

What are the components of PCR and what do they do?

• Template: the DNA from the sample specimen serves as the template for replication • Primers: short stretches of DNA that initiate the PCR reaction. • Required for annealing and Taq binding at specific sites • Base sequences are complementary to the ends of the template DNA • Taq polymerase: adds dNTPs to the template strand • Reads the original DNA sequence and creates a complimentary copy by adding in the new DNA bases • Nucleotides (dNTPs): DNA bases (A, C, G, and T) serve as the building blocks of DNA and are used to assemble new strands of DNA • Buffer: enables the reaction to take place by ensuring the right conditions are met (controls pH)

What does the chelex do?

The chelex resin chelates (absorbs) ions that inhibit function of the Taq polymerase enzyme

What does the thermocycler do?

Thermocyclers are instruments used to amplify DNA and RNA samples by the polymerase chain reaction. The thermocycler raises and lowers the temperature of the samples in a holding block in discrete, pre- programmed steps, allowing for denaturation and reannealing of samples with various reagents.

What are the 3 steps of a PCR cycle? What happens during each step?

• 1) Denaturation, 2) Annealing, 3) Extension • Denaturation: When the double stranded template DNA is heated to separate it into two single strands • Annealing: when the temperature is lowered to enable the DNA primers to attach to the template strands • Extension: when the temperature is raised and the Taq polymerase enzyme synthesizes new DNA strands by adding nucleotides to the template strands.

• What is the purpose of lysing the DNA?

Lysis destroys the cell membrane and releases the contents of the cell into the solution

What organism was Taq polymerase isolated from?

Thermus aquaticus: thermophilic bacterium (heat tolerant) that live in hot springs

What is multiplex PCR?

Amplification of multiple targets into a single PCR experiment by utilizing multiple primer pairs in a single reaction mixture

What is the purpose of gel electrophoresis?

Allows you to separate a mixture of different sized DNA molecules so that DNA samples can be compared to other known DNA profiles.

What is the charge of DNA?

DNA has a negative charge

What direction should DNA run?

Toward the positively charged end (anode)

What are things that influence how far DNA will run?

Charge of molecule: DNA carries a negative charge so attracted to positive end • Size: Smaller molecules/fragments will travel faster and further • Density of gel: the more dense the gel is, the slower the particles will move. You want a gel that is dense enough to separate particles but that won't take too long

Who will run further from a well? 700bp of DNA or 200bp of DNA.

200 bp (smaller, so travels faster and further)

Why is developing a DNA profile important?

DNA testing allows forensic investigators to identify a guilty individual with a high degree of certainty because the DNA sequence of every person is unique (except for identical twins)

How did multiplex PCR help in our analysis versus just using a single pair of primers?

A single pair would not allow you to distinguish between samples/individuals with a high degree of certainty

Why are DNA ladders (standards) important?

They are used to determine the sizes of bands in other DNA samples. Thus, we are able to compare them.

Can you tell the difference between a homozygote or heterozygote gene on a gel?

Yes, a homozygote will have one band at a particular loci and a heterozygote will have two bands at a particular loci

What was the purpose of using SYBR Green in the gels?

SYBR Green binds to DNA and fluoresces when exposed to the right wavelength of light. This makes the separated bands visible.

What was the purpose of pouring the buffer in the electrophoresis chamber?

Buffer controls the net charge of molecules by maintaining the pH at nearly neutral. This facilitates proper migration and separation of DNA molecules.

What is the central dogma of gene expression? Define transcription and translation.

The central dogma states that genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins o Transcription: DNA from a gene sequence is copied to mRNA o Translation: directs the synthesis of proteins

What is the name of the primary enzyme used in this lab? Where is it coming from?

B-galactosidase is an enzyme is E. coli that is encoded by the LacZ gene when E. coli needs to break down lactose

What are the components of the lac operon and what do they do?

Operon: is a functioning unit of DNA containing a cluster of genes under the o RNA polymerase: needed to start transcription and produce mRNA o Promoter: the binding site for RNA polymerase o Operator: negative regulatory site that the repressor can bind to o CAP site: positive regulatory site that catabolite activator protein (CAP) can bind to o Repressor: when the repressor is bound it blocks RNA polymerase and prevents the production of mRNA (so no production of proteins) o The lac operon has 3 genes that help the cell utilize lactose: lacZ - encodes β-galactosidase enzyme, which splits lactose into monosaccharides. lacY - encodes lactose permease protein, which is a transmembrane "pump" that allows the cell to import lactose. lacA - encodes transacetylase enzyme

E. coli makes B-gal to breakdown what sugar?

Lactose

Expression of the LacZ gene causes the production of what enzyme?

B-gal

How can ONPG be used to measure optimal growth conditions for the expression of LacZ?

Because B-gal also breaks down ONPG and one of the products of this reaction has a yellow color (ortho-nitrophenol), we can investigate the regulation of LacZ transcription by observing the rate of color change in the tubes. Presence of yellow color would mean that B-gal was produced, thus LacZ would be expressed.

How does LacZ/B-gal expression influence activity? If LacZ/B-gal expression is high/low, how quickly do you think ONPG will be broken down into galactose and o-nitrophenol?

If LacZ/B-gal expression was high, ONPG would be broken down quickly because there would be more enzymes available.

Understand how LacZ expression changes when E. coli is grown under different culture conditions: LB (plain media), LB+lactose, LB+glucose, and LB+glucose+lactose. Is the activity predicted to be high or low?

Activity would be highest in the LB+lactose culture

calculate Activity.

Activity = Absorbance/time * 1000 (MUST multiply by 1000!!!)

Understand how adding sugars to the B-gal assay influences activity.

Adding the sugars in Activity 2 should have had little to no effect on the assays. The more important factor affecting expression was which sugar was present INSIDE the culture. Otherwise, we would have expected the LB culture + lactose solution to have the most activity.

What sugar is similar to ONPG?

Lactose

What sugars are the products of the hydrolysis of lactose?

Glucose and Galactose

In what sugar conditions is the repressor bound or unbound to the operon?

The repressor is bound to the operator when lactose is absent. When lactose is present, allolactose binds to the repressor, which prevents the repressor from binding to the Lac operon's operator

In order to make more B-gal, what will the repressor bind?

If B-gal needed to be produced, allolactose would have to bind to the repressor

Know the magnification of each lens and how to calculate total magnification.

Objective lens: 4X, 10X, 40X, 100X (100X is only for oil immersion) o Ocular lens: 10X magnification o Objective magnification + ocular magnification = total magnification. 4X 10X = 40X total magnification § 10X 10X = 100X total magnification. 40X * 10X = 400X total magnification

Describe how you would use a compound microscope starting with the 4X objective lens and ending with the 40X objective lens.

With the 4X objective lens you would first focus your slide using the coarse adjustment knob, then the find adjustment knob until the image is as clear as you can get it. Then you would move to the 10X objective lens and refocus using only the FINE adjustment knob. Then you would move up to the 40X objective lens and refocus using only the FINE adjustment knob.

Know the stages of the cell cycle and phases of mitosis. Be able to briefly summarize what happens at each stage. Also, be able to visually identify each stage.

Interphase: cell growth and DNA replication (G1, S, and G2 phases). G1 phase: period of intense growth and biochemical activity. S phase: synthesis/replication of DNA. G2 phase: cell continues to grow and complete preparations for cell division. Mitosis: is a continuous process and consists of the actual dividing of the nucleus (replicated DNA and cytoplasmic contents are separated) Prophase: nuclear membrane begins to disintegrate and the chromosomes begin to condense. Metaphase: chromosomes line up in center of cell. Anaphase: centromeres of each chromosome separate, as spindle fibers pull apart the sister chromosomes. Telophase: chromosomes cluster at opposite ends of the cells, and the nuclear membrane reforms. Chromosomes start to unravel. Cytokinesis: consists of the dividing of the cytoplasm. Cleavage furrow forms in animal cells and cell plate forms in plant cells.

Be able to figure out how many chromosomes are at each stage of interphase and mitosis.

Mitosis produces two genetically identical daughter cells and conserves the number of chromosome number (2n). If a cell starts with 46 chromosomes in interphase, then there would be 46 chromosomes at prophase and metaphase, 92 chromosomes in anaphase and telophase, a

Know what the difference is between mitosis and meiosis.

Mitosis produces two genetically identical diploid daughter cells and conserves the number of chromosome number (2n). Genetic variation does not change. Meiosis occurs in sexually reproducing organisms and results in cells with half the chromosomes number of the parent cell (n). Meiosis produces 4 haploid daughter cells and increases genetic variation.

Why did we only want to look at the root tips to observe the cell cycle?

The apical meristem is located in root tips. This is a region of rapid progression through the cell cycle to increase the number of cells.

What are the plant mutants that we worked with in class? What are their mutant phenotypes?

We worked with Arabidopsis thaliana, which is a small weed in the mustard family and is a common model organism for studying plants (bc of it's small size, rapid generation time, and ease of growing in labs) o One parent had trichomes and fluoresced (hair + glow) o One parent was glabrous and did not fluoresce (no hair + no glow)

What is a phenotype?

The physical characteristics of a trait (i.e., what it looks like - color, texture, size, etc.)

What is a genotype?

The particular combination of alleles for a gene or locus i.e., The letter combination that makes up a trait (ex. AA, Aa, aa)

Homozygous

two identical genes for the same trait (ex. AA or aa) Homozygous dominant: contains two dominant genes/alleles (ex. AA, BB) Homozygous recessive: contains two recessive genes/alleles (ex. aa, bb)

Heterozygous

two different genes for the same trait. Contains one dominant gene/allele and one recessive gene/allele (ex. Aa/Bb)

Allele

a gene for a particular trait (ex. A or a)

Gene

the segment of DNA that determines a particular trait

Trait

an inherited characteristic of an organism

Law of Dominance:

when two organisms that are each homozygous for two opposing traits are crossed, the offspring will be hybrid (carry two different alleles) but will exhibit only the dominant trait. The trait that remains hidden is the recessive trait.

Law of Segregation:

states that during the formation of gametes, the two traits carried by each parent will separate. § The cross that best exemplifies this law is the monohybrid cross (Tt x Tt), where a trait that was not evident in either parent appears in the F1 generation

Law of Independent Assortment

states that during gamete formation, the alleles of a gene for one trait, such as height (Tt), segregate independently from the alleles of a gene for another trait such as seed color (Yy). This law applies when a cross is carried out between two individuals hybrid for two or more traits that are not on the same chromosome (dihybrid cross)

Monohybrid cross (Tt x Tt):

is a cross between two organisms that are each hybrid for one trait. The phenotype (appearance) ratio from this cross is 3 tall to 1 dwarf plant. The genotype ratio is 1:2:1.

Dihybrid cross (TtYy x TtYy):

is a cross between individuals that are hybrid for two different traits. This cross can produce four different types of gametes (TY, Ty, tY, ty). The phenotypic ratio of a dihybrid cross is 9:3:3:1

What is bioinformatics?

The application of computer technology and associated software to biological data

What does NCBI stand for? What is NCBI used for?

National Center for Biotechnology Information (NCBI) • NCBI is a database that stores sequence information, taxonomic data, 3D protein structures, scientific literature, and a vast array of both online and offline software tools.

What does BLAST stand for? What is BLAST used for?

Basic Local Alignment Search Tool (BLAST) • BLAST is used to generate alignments between a nucleotide or protein sequence between other sequences in the database (in order to identify organisms, find out the function of a gene or protein, etc.)

What is the difference between BLASTn and BLASTp?

BLASTn compares nucleotide sequences • BLASTp compares protein sequences

What is the purpose of a phylogeny?

To group organisms by similarity (i.e., species, genes, proteins, morphology, etc.)

What is the max score?

Max score is based on the length of the query sequence matched. It is the highest alignment score calculated from the sum of matched and mismatched nucleotides or amino acids.

What is the percent sequence identity?

The percent identity tells you how many characters (bases or amino acids) match exactly between the query sequence and the target sequence

What is an E-value?

The E-value represents the probability that you would get a match with that score by random chance (the number of expected hits of similar quality that could be found by chance)