Genetics Exam 4 SLOs

Uses of PCR

amplify (make copies of) DNA sequence, prepare DNA for cloning, detect presence of specific genes (pathogens and mutations), research

Steps in PCR

1. Denaturation → heating, two strands separate to one

2. Annealing → primers bind to complementary target sequence

3. Extension → DNA polymerase extends primers → synthesize new DNA strands

Uses of RT-PCR

gene expression, RNA→DNA for amplification, detect RNA viruses, create cDNA (important for cloning expressed genes)

Steps of RT-PCR

1. Reverse Transcription → RNA converted to complementary DNA (cDNA) using reverse transcriptase

2. PCR Amplification → cDNA amplified using PCR

Key Difference in PCR and RT-PCR

PCR → starts with DNA

RT-PCR → starts with DNA → cDNA first

Library Cloning

collection of many DNA fragments (DNA library) that must be screened to find gene of interest

Specific Fragment Cloning

isolates and amplifies single known DNA sequence directly, often w/ PCR, no screening needed

Key properties of cloning vectors

origin of replication (ori)

multiple cloning site (MCS)

selectable marker

screenable marker

Origin of replication (ori)

allows vector to replicate in host cell

Multiple Cloning site (MCS)

contains restriction enzyme sites for inserting DNA

Selectable Marker

identifies cells that took up vector (e.g. antibiotic resistance)

Screenable Marker

distinguishes between cells with different plasmids (with and without inserts); helps you tell which cells have the desired insert, without killing any cells

Action of restriction enzymes

Recognizes specific DNA sequences → usually palindromic sites

Cuts DNA at those sites → break DNA into fragments

Creates sticky or blunt ends → determines how fragments are joined

Cuts both insert and vectors → ensures compatible ends

Allows ligation → fragments can be joined together by DNA ligase

Predict average size of fragments from given restriction digest

• Find recognition site length (n) → count number of bases (e.g., 4 bp, 6 bp)

• Assume equal base frequency → each base = 1/4 chance

• Calculate average spacing → fragments occur every 4ⁿ bases

• Result = average fragment size

EXAMPLE: 4-base cutter → 4⁴ = 256 bp 6-base cutter → 4⁶ = 4096 bp

SHORT: Average fragment size = 4ⁿ, where n = length of the recognition site.

Given unfamiliar plasmid vector, choose appropriate restriction site into which to place an insert

Pick a unique restriction site in the MCS that doesn’t disrupt essential genes and has compatible ends with your insert

PICK SITE in multiple cloning site (MCS) → designed for DNA insertion

Avoid essential regions → DO NOT cut origin of replication or selectable marker

Preferred sites within screenable marker (e.g. lacZ) → allows identification of inserts

Ensure compatible ends → match insert and vector (sticky or blunt)

Use unique restriction sites → cut plasmid only once to prevent fragmentation

Describe CRISPR cloning

• Identify target gene → decide what DNA to edit

• Design guide RNA (gRNA) → directs Cas9 to specific DNA sequence

• Cas9 cuts DNA → creates a double-strand break at target site

• Provide donor DNA (optional) → contains desired insert or mutation

• DNA repair occurs →

  • NHEJ → may create mutations (indels), quick, error prone,

  • HDR → inserts desired sequence using template

• Screen/select cells → identify successful edits

CRISPR cloning uses gRNA and Cas9 to cut DNA at a specific site, then cell repair inserts or modifies the sequence. CRISPR doesn’t just cut DNA—it uses the cell’s repair system to either disrupt a gene (NHEJ) or precisely insert new DNA (HDR).

CRISPR: Cell tries to repair the break → two main pathways

• NHEJ (non-homologous end joining)

  • quick, error-prone

  • often creates small insertions/deletions → can knock out a gene

• HDR (homology-directed repair)

  • uses a donor DNA template you provide

  • allows inserting or changing a specific sequence (true “cloning/editing”)

Southern Blot

Detects specific DNA; tells you if a gene is present, its size, and copy number (Specificity comes from labeled DNA probe binding to a complementary DNA sequence)

Binding is sequence specific

Probe: gene in question

Northern Blot

detects RNA, tells you if a gene is expressed and how much mRNA is made (probe binds to specific RNA sequence)

Binding is sequence specific

Probe: gene transcript in question

Western Blot

detects proteins using antibodies, tells if protein is present, size, and expression level (antibodies binding to specific protein)

Binding is antibody specific

Probe: antibody to protein in question

Main steps of negative transcriptional regulation

(repressor)

1. repressor protein binds DNA at operator→ blocks transcription (effector that makes the repressor protein get on DNA is co-repressor)

2. Prevents RNA polymerase binding or movement

3. Gene is OFF when repressor is bound

4. Removal/inactivation of repressor (effector that makes the repressor protein get off DNA is inducer - inducible operon, effector that makes the repressor get on the DNA is a co-repressor and repressible operon)→ transcription ON

Main steps of positive transcriptional regulation

(activators)

1. Activator protein binds DNA before operator site(called activator)→ helps transcription

2. Recruits or stabilizes RNA polymerase

3. Gene is ON when activator is bound

4. Without activator → low or no transcription

Lac Operon

a set of genes in bacteria (e.g., E. coli) responsible for breaking down lactose; remaining "off" when lactose is absent to save energy, and turning "on" only when lactose is available and glucose (the preferred energy source) is absent. Regulated by single promotor (P) and operator (O)

Lactose will bind the repressor, changing its shape so it cannot bind the operator, transcription ON

Repressor protein binds operator, blocking transcription

Going through the Lac operon chart

1. Check both P’s, if mutated, then cross off that operon (won’t be expressed)

2. Check both O’s, if mutated, operon will be constitutively expressed

3. Check both I’s to determine if regulation will be inducible (normal), uninducible (no expression), or constitutive

4. Assess Z and Y to see the alleles are wt (wild type) or mutant (inactive even when expressed)

REMEMBER PROTEINS ARE TRANSACTING WHILE DNA SITES ARE CIS-ACTING

When should you ignore lactose?

• Oᶜ → already ON

• P⁻ → already OFF

• Iˢ → always OFF

• I⁻ (no I⁺ around) → always ON

Reading Lac Operon Chart: I (affects both copies)

I+: wild-type repressor gene; active and binds to the operator (O) in absence of lactose preventing transcription of lacZ (beta-galactosidase) and lacY (permease) genes; means the operon is inducible (off without lactose, on with lactose

I- : Constitutive Expression (Always On); defective repressor protein that cannot bind to the operator (O)

I^s: Super-Repressor, cannot bind to the inducer (non inducible); "permanently off" phenotype for both β-galactosidase (encoded by lacZ) and permease (encoded by lacY).

Reading Lac Operon Chart: P

P+: wild-type (functional) promoter; allows it to initiate transcription of the downstream;
cis-acting → operates on the same piece of DNA that it is physically linked to; alongside a functional repressor (I+) and operator (O+), the genes are produced only when lactose is present

P- : : Defective promoter (transcription cannot occur); cannot produce β-galactosidase or permease, regardless of whether lactose is present or absent; cis-acting → only affects DNA piece it’s directly attached to (cannot be fixed)

Reading Lac Operon Chart: O (cis-acting)

O+ : operator is wild type; normal and can bind to the repressor protein produced by the regulator gene; If no lactose is present resulting in negative (-) enzyme production; If lactose is present, resulting in positive (+) enzyme production

Oc: Operator-Constitutive; prevents the Lac repressor protein from binding; constantly "ON" or produced regardless of whether lactose is present or absent

Reading Lac Operon Chart: Z

Z+ : wild type; possesses the genetic information to produce active, functional β-galactosidase enzyme; gene can be expressed when the operon is "on"; tells you that if transcription occurs

Z- : mutation that leads to nonfunctional or no β-galactosidase; enzyme responsible for breaking down lactose into glucose and galactose is absent or defective; cannot utilize lactose

Reading Lac Operon Chart: Y

Y+ : wild type (functional); produces functional lactose permease, a membrane protein that allows lactose to be transported from the environment into the cell; inducible, meaning the permease is only produced when lactose is present

Y- : means the gene is non-functional or mutant, resulting in a loss of function for the membrane-bound transport protein; cannot effectively transport lactose; Non-functional permease / No lactose uptake.

Map of Lac Operon

POZY

Promoter, operator, ZYA

Allolactose

natural inducer of the lac operon in E. coli, initiating gene expression for lactose metabolism. It is formed from lactose via β-galactosidase and binds to the lac repressor

How does glucose influence the Lac Operon

High glucose keeps cAMP levels low, keeping transcription low. Low glucose leads to high cAMP, which activates the CAP protein to enhance transcription.

LacA

transacetylase; encodes β-galactoside transacetylase; transfer an acetyl group from acetyl-CoA to galactosides

LacZ

β-galactosidase; encodes the enzyme β-galactosidase; cleave the disaccharide lactose into its constituent monosaccharides: glucose and galactose, which can then be used in glycolysis for energy

LacY

lactose permease; encodes a transmembrane protein β-galactoside permease, or lactose permease. This protein acts as a pump, inserting into the cell membrane to facilitate the transport of lactose from the environment into the cell

Using PCR & blotting to study an unfamiliar gene

• PCR → amplify specific regions → identify gene sequence or structure

• RT-PCR → detect mRNA → determine if the gene is expressed

• Southern blot → analyze DNA → gene presence, size, or copy number

• Northern blot → analyze RNA → transcript size and expression level

• Western blot → analyze protein → confirms protein production and size

• Compare all levels → DNA → RNA → protein to understand gene function

Use PCR to amplify the gene and blots (Southern, Northern, Western) to analyze it at the DNA, RNA, and protein levels.

Main steps of transcriptional regulation in eukaryotes

1. Chromatin remodeling (before transcription) → DNA is made accessible (euchromatin) or inaccessible (heterochromatin)

2. Epigenetic modifications → DNA methylation and histone modifications regulate gene activity

3. Transcription factor binding → activators and repressors bind to promoters, enhancers, and silencers

4. Recruitment of RNA polymerase II → transcription machinery assembles with help of coactivators

5. Initiation of transcription → RNA synthesis begins if conditions are favorable

6. Combinatorial Control → different combinations of transcription factors determine gene expression levels

Main steps of post-transcriptional regulation in eukaryotes

1. Alternative splicing → different exon combinations, produce different mRNAs/proteins

2. RNA processing/modifications → 5’ cap and poly A tail added for stability and translation

3. RNA interference (RNAi) → small RNAs (miRNA/siRNA) bind mRNA to block translation or cause degradation

4. mRNA stability/degradation → lifespan of mRNA determines how much protein is made

5. Transcriptional Region → mRNA can be blocked or activated for translation

Histone Modification

adding acetyl groups to histones open chromatin → increasing transcription

DNA methylation

methyl groups added to DNA (CpG sites → cytosine followed by a guanine) → decreasing transcription

Epigenetics

heritable changes in gene expression without changing DNA sequence (e.g. X-chromosome inactivation)

Transcription initiation

transcription factors bind promoters/enhancers to start or block RNA synthesis

mRNA processing

alternative splicing creates different mRNAs from same gene

Translation

miRNA binds mRNA to block protein production

Protein modification

proteins are activated/inactivated after translation (e.g. phosphorylation)

Using a codon table to assess mutations

• Identify the DNA or mRNA sequence → determine the original codons

• Apply the mutation → change the nucleotide(s) given

• Convert to codons → group into sets of three bases

• Use codon table → determine the amino acid(s) before and after mutation

• Compare results: what type of mutation?

• Predict effect on protein → little, moderate, or major impact depending on change

Silent mutation

no amino acid change, Occurs because the genetic code is redundant (multiple codons code for same amino acid)

• Usually no effect on protein function

• Original: AAA → Lysine Mutated: AAG → Lysine (same amino acid → silent)

Missense Mutation

Different amino acid; Caused by a single base substitution

• Can have no effect, mild effect, or severe effect depending on the amino acid change

• (Original: GAA → Glutamic acid Mutated: GUA → Valine)

Nonsense Mutation

Premature stop codon formed; early termination of translation

• Results in a shortened (truncated), usually nonfunctional protein

• (Original: UAU → Tyrosine Mutated: UAA → Stop)

Frameshift (specific sequences of indel) Mutation

shifts reading frame, changes many amino acids; Insertion or deletion of nucleotides (not in multiples of 3); Changes all downstream amino acids

• Often produces a nonfunctional protein

• (Original: THE CAT ATE... After deletion: THC ATA TE… → completely different message)

Indel

insertion or deletion of nucleotide(s) in DNA, Can be small (1 base) or larger segments, If not in multiples of 3 → causes a frameshift mutation

Genetic Analysis using Mutagenesis

1. DEFINE GOAL → what gene/process

2. CHOOSE DESIRE MUTANT TYPE → loss of function, conditional

3. SELECT APPROPRIATE MUTAGEN → base analogs/alkylating agents, intercalating agents, UV light

4. Mutagenize organism → expose cells to mutagen

5. Screen/select mutant → identify individuals with desired phenotype

6. Analyze result → connect mutation to function

Choose a mutant type, apply a mutagen to create mutations, then screen for desired phenotypes and analyze how the mutation affects the biological process.

Mutagenesis

cell’s DNA sequence is changed, creating mutations; genetic code gets altered, either naturally or artificially

TYPES: Spontaneous mutagenesis and Induced mutagenesis

Induced mutagenesis

Caused by external factors called mutagens, such as:

• UV radiation (sunlight)

• Chemicals (like certain pollutants or lab reagents)

• Ionizing radiation (X-rays)

Spontaneous mutagenesis

Happens naturally due to errors in DNA replication or normal cell processes.

Loss-of-function (null)

causes a gene product (usually a protein) to lose its normal activity or function, protein may be:

• Not made at all

• Made but nonfunctional

• Made but less active than normal

Example idea: A mutation in an enzyme gene that stops the enzyme from working → a metabolic pathway is disrupted.

Conditional Mutation

only shows its effect under certain environmental conditions.

• The organism appears normal under “permissive” conditions

• But shows a mutant phenotype under “restrictive” conditions

Example idea:

• A bacterium grows normally at 30°C (permissive temperature)

• But cannot survive at 42°C (restrictive temperature)

base analogs/alkylating agents

Mimic normal DNA bases and get incorporated into DNA

Cause mispairing (often via tautomeric shift - temporary change in the chemical structure of a DNA base or base analog, pairs incorrectly during DNA replication) → Base substitution mutations (mispairing during replication)

Alkylating Agents

Chemically add alkyl groups to DNA bases/modify bases → alter pairing properties →

Main effect: Base mispairing, base damage, sometimes strand breaks, substitutions

intercalating agents

Insert between DNA adjacent base pairs (distort DNA structure) → cause replication indel/slippage → insertion/deletions (frameshift)

• During replication, DNA polymerase may:

  • Insert extra bases (insertion mutations)

  • Skip bases (deletion mutations)

(physically “stuck themselves” into DNA, causing copying errors)

UV light/radiation

Forms thymine dimers (two thymine adjacent bond instead of normal pairing, distorts DNA helix) → blocks replication → leads to base damage and incorrect repair (permanent)→ replication errors (small-scale mutations)

(SKIN CANCER)

Ionizing radiation (X-rays, gamma rays)

Break DNA strands directly or indirectly via free radicals → error prone repair → mutations

Single- and double-strand breaks → large chromosomal damage

Hypomorphic Mutation (LEAKY)

partial function → milder phenotype

Hypermorphic Mutation

Increase activity → exaggerated phenotype (expressed more)

Gain-of-Function Mutation

new/abnormal function → can be harmful or novel effect

Direct Repair of DNA Damage

• Reverse damage directly → fixes altered base without removing it

• No DNA replacement needed

• Specific to certain damages (UV light, alkyl groups)

• Quick and accurate (in place repair)

Excision Repair (cut out and replace) of DNA Damage

• Damaged DNA removed → base, nucleotide, or short segment is cut out

• DNA polymerase fills in correct nucleotides using opposite strand as template

• DNA ligase seals strand

• Includes → base excision repair, nucleotide excision repair, and mismatch pair

• Specific damage → Alkylated bases, UV-induced thymine dimers,

Base excision repair (BER) and Nucleotide excision repair (NER)

• Base excision repair (BER) — small, single-base damage, Fixes damage to individual bases that don’t significantly distort the DNA helix

• Nucleotide excision repair (NER) — bulky, helix-distorting damage, Fixes larger lesions that distort the DNA structure

BER → fixes single-base chemical damage

NER → fixes bulky, helix-distorting damage (like UV dimers)

Plasmid

small insert, uptake by transformation, easy and cheap, used in genomic library, cDNA library, subcloning

Phage

medium insert, uptake by transduction (infection), easy, used in subcloning and mutagenesis

Cosmid

large insert, uptake by transduction (infection - most efficient way of getting into cell), hard, used in large scale sequencing/mapping

BAC

Bacterial artificial chromosomes; low copy number (one to two copies per cell) huge, uptake by transformation, tough, used in whole genome sequencing

YAC

yeast artificial chromosome; have telomeres at each end, ORI, centromere; gigantic, uptake by transformation, very tough, used in whole genome sequencing

Expression Vectors

ensures mRNA expression of cloned gene - to produce large quantities of encoded protein in host cell

plasmids, phage vectors, and YACs only carry DNA and don’t signal for protein

Shuttle vectors

Allow for replication and expression in E. coli and another. species

Transition Mutations

point mutation where a purine base is swapped for another purine (A ↔ G) or a pyrimidine for another pyrimidine (C ↔ T). These mutations are more frequent in genomes than transversions because they are less disruptive to DNA structure and are often caused by spontaneous tautomerization or deamination

Transversion Mutation

point mutation in DNA where a purine base (Adenine, A; Guanine, G) is replaced by a pyrimidine base (Cytosine, C; Thymine, T), or vice versa; change the purine-pyrimidine structural pairing, making them more likely to alter protein function or local DNA structure

Somatic Mutation

acquired changes in non-reproductive cells post conception

Germline Mutation

Mutations inherited changes in reproductive genes

Spontaneous Mutation

occur naturally, often during DNA replication

Induced Mutation

Cause by external environmental factors called mutagens (forced by chemical agents)