Microbiology exam 2

Point mutation

A single-base change in the DNA sequence.

Frameshift mutation

Insertion or deletion of nucleotides, causing a shift in the reading frame.

Forward mutation

Changes a wild-type gene to a mutant phenotype.

Back mutation

Reverses an earlier mutation at the same site.

Suppressor mutation

Compensates for the effects of a previous mutation at a different site.

Silent mutation

A mutation that doesn't alter the encoded amino acid.

Know the difference between a transition and a transversion, including which is more common

Transition: Purine-to-purine or pyrimidine-to-pyrimidine substitution.

Transversion: Purine-to-pyrimidine or pyrimidine-to-purine substitution.

Transitions are more common than transversions.

Name two types of frameshift mutation

Insertion: Adds nucleotides, shifting the reading frame.

Deletion: Removes nucleotides, shifting the reading frame.

Compare and contrast back mutation and suppressor

Back mutation reverses the original mutation at the same site, while suppressor mutation compensates for the original mutation at a different site.

Understand why a neutral substitution is considered a silent mutation

A neutral substitution doesn't change the amino acid sequence due to redundancy in the genetic code, making it silent at the phenotypic level.

Genome

The complete set of genes present in an organism's DNA.

Transcriptome

The complete set of RNA molecules transcribed from the genes in a cell, tissue, or organism.

Proteome

The complete set of proteins encoded by the genome or produced by a cell, tissue, or organism.

Ortholog

Genes in different species that evolved from a common ancestral gene by speciation.

C-value

The total amount of DNA in a haploid genome.

Know the estimated number of genes encoded in the human genome

Approximately 20,000 genes

Describe the difference between repetitive and nonrepetitive genomic DNA

Nonrepetitive DNA consists of unique sequences that appear only once in the genome, typically encoding protein-coding genes.

Repetitive DNA consists of sequences that appear multiple times in the genome, classified as moderately repetitive (10-1,000 times) or highly repetitive (1,000s of times).

Discuss if (1) genome size and (2) organism complexity are correlated to the number of genes in eukaryotes

Genome size (in base pairs) does not always correlate with the number of genes.

There is a positive correlation between organism complexity and the number of genes.

Explain the C-value paradox

Lack of a clear relationship between genome size (C-value) and organism complexity. Despite variations in genome size, organisms of similar complexity may have vastly different amounts of DNA.

GWAS

Genome-wide association studies (GWAS) examine the entire genome to find genetic variations associated with traits or diseases.

Polymorphism

The presence of multiple alleles at a specific locus in a population.

SNP

Single nucleotide polymorphism (SNP) is a variation in a single nucleotide base in DNA.

Explain why SNPs are essential to perform GWAS

SNPs serve as genetic markers, helping researchers identify regions of the genome linked to traits or diseases

Know what data from GWAS can tell you (and what it can’t)

GWAS can identify associations between specific SNPs and traits or diseases, but it cannot directly pinpoint the specific genes involved

Understand the potential clinical impacts of GWAS

GWAS can lead to the discovery of new diagnostic markers and personalized treatment approaches based on individuals' genetic profiles.

Origin of Replication

The site on a chromosome where DNA replication begins.

Replication Fork

A Y-shaped structure formed during DNA replication where the double helix is unwound and new DNA strands are synthesized.

RNA Primer

A short chain of RNA that initiates DNA replication by providing a free 3’ -OH end for DNA polymerase to add nucleotides onto

DNA Polymerase

An enzyme responsible for synthesizing new DNA strands by adding nucleotides in a complementary fashion to the template strand

DNA Helicase

An enzyme that unwinds and separates the DNA double helix during replication

DNA Primase

An enzyme that synthesizes RNA primers needed to initiate DNA replication

DNA Ligase

An enzyme that joins Okazaki fragments on the lagging strand during DNA replication

Topoisomerase

An enzyme that relieves the tension caused by DNA unwinding during replication by making small cuts in the DNA strands

SSB Proteins

Single-strand DNA-binding proteins that stabilize single-stranded DNA during replication, preventing them from reannealing.

Semi-conservative Replication

The mechanism of DNA replication where each parental strand serves as a template for the synthesis of a new complementary strand

What allows DNA replication to happen quickly in our cells?

Replication can be fast because it is semi-conservative, initiated at multiple origins, and proceeds bidirectionally from each origin

What are the molecular events during the initiation, elongation, and termination phases of replication?

Initiation involves DNA helicase unwinding DNA, single-strand DNA-binding protein keeping strands separate, and topoisomerase relieving tension. Elongation includes DNA polymerase synthesizing DNA, exonuclease activity proofreading, and DNA ligase sealing fragments. Termination involves completion of replication

How and why does DNA replication differ on the leading and lagging strands?

The leading strand is made continuously in the 5’ to 3’ direction, while the lagging strand is made discontinuously in short fragments (Okazaki fragments) in the 3’ to 5’ direction

What is the Telomere Problem and its biological solution?

The Telomere Problem refers to the shortening of telomeres during replication, leading to cell aging. Telomerase is the enzyme that replicates telomeres, preventing their shortening and enabling cells to continue dividing.

List the (5) components of a PCR and what they do in the reaction

1. DNA Template: Target DNA sequence for amplification.

2. Primers: Bind to flanking sequences for DNA synthesis initiation.

3. dNTPs: Building blocks for DNA synthesis.

4. DNA polymerase: Catalyzes DNA strand synthesis.

5. Buffer (+ Mg2+): Provides optimal reaction conditions.

Explain what happens during the denaturation, annealing, and extension steps of a PCR cycle

Denaturation: 95°C for 15-30 seconds

Annealing: 50-65°C for 30-60 seconds

Extension: 72°C for 1 minute/kb

Compare and contrast PCR and DNA replication

PCR: Amplifies specific DNA sequence, uses synthetic primers, occurs in vitro

DNA replication: Copies entire genome, uses RNA primers, occurs in vivo

recombinant DNA

artificial DNA molecules made by combining 2+ pieces of DNA from different sources

vector

plasmid used to replicate a cloned DNA segment

insert

fragment of DNA that is to be cloned

restriction enzyme

endonuclease that ‘cuts’ a specific DNA sequence

blunt end

a DNA cutting result where both strands of the DNA molecule are cleaved at the same point, producing ends with no overhanging nucleotides, resulting in a flat or blunt terminus.

sticky end

DNA cut, leaves overhang, can bind with complementary sequence.

multiple cloning site (MCS)

Region with many unique restriction sites (cut sites)

•Allows ‘easy’ insertion of gene of interest

Know the function of the ori

Allows plasmid to replicate in the host

Know the function of Selection markers

Gene that allows cell to survive under specific selection conditions

•Ex. antibiotic resistance gene

Allows you to pick out bacteria that carry your vector

Know the function of MCS in a cloning vector

Region with many unique restriction sites (cut sites)

Allows ‘easy’ insertion of gene of interest

Explain how you can verify your gene of interest was successfully incorporated into a vector

Transform cells, plate on antibiotic media.

White colonies indicate successful ligation (recombinant plasmid), blue colonies indicate unsuccessful ligation (non-recombinant plasmid).

Understand what you can study ask using (1) an expression vector and (2) a reporter gene

1. Produces proteins for commercial use

2. Studies promoter activity in vivo.

Discuss several ways vectors can be introduced into different target cells

Heat shock, electroporation, chemical transformation.

DNA profiling

Analysis of specific DNA regions to create unique genetic fingerprints.

minisatellite

DNA sequences with repeats of 10bp – 1000bp

Microsatellite

DNA sequences with repeats of 2-10bp

Explain why microsatellites/minisatellites are useful in DNA profiling

Variation in repeat number creates unique genetic profiles.

Enables accurate identification and differentiation between individuals.