BIO EXAM 2 (2nd half)

Small genetic changes (base pair changes, gain of genes) can be

beneficial

Evolution of species adapt to their environment through

gradual genetic changes, ex. peppered moth changed from “peppered” to black during the Industrial Revolution in the 1800s when soot blackened the trees → camouflage from birds when environment changed

Many genetic changes have no effect

“silent” mutation

Silent mutations happen…

• in non coding regions of DNA (regions that do not code for genes)

• in gene but does not change the protein sequence because of the redundancy in the genetic code (i.e. multiple nucleotide sequences code for the same amino acid)

Examples of how genetic changes can be harmful

• Can cause diseases → cancer

• Inheritable genetic disorders such as sickle cell anemia, huntingtons disease, and cystic fibrosis

Cystic fibrosis can arise from multiple mutations but the most common is

a deletion of three nucleotides from the cystic fibrosis transmembrane regulator gene resulting in the loss of one amino acid (phenylalanine) in the protein

The majority of DNA damage comes from

• Reactive chemicals produced in cell metabolism and chemical reactions in the cell alter nucleotide

• Environmental factors/agents

  • UV light

  • Radiation

  • Toxic chemicals

Some mutations arise during DNA replication due to

mismatches, proofreading ability of DNA polymerase corrects these in most cases

How does DNA polymerase proofread newly synthesized DNA?

DNA polymerase has two active sites:

• Polymerizing site (P): adds nucleotides to the growing DNA strand

• Editing site (E): removes incorrectly added nucleotides

The enzyme initially binds DNA loosely and must undergo a conformational change to form a phosphodiester bond—this helps many incorrect nucleotides fall off before being added.

If a wrong nucleotide is incorporated, the DNA briefly unpairs, and the 3′ end shifts to the editing site, where the incorrect nucleotide is removed (exonuclease activity), allowing synthesis to continue correctly.

Why is DNA synthesized only in the 5' to 3' direction?

Synthesis in the 5' to 3' direction allows for proofreading; if an incorrect nucleotide is removed, the incoming correct triphosphate provides the high-energy bond needed to restart polymerization, which would be impossible in the 3' to 5' direction.

What happens to a DNA mismatch if it is not corrected before the next round of replication?

It becomes a permanent mutation, because once the mismatched strand serves as a template, the incorrect base is paired with its complementary partner, making it indistinguishable from the original sequence.

Protein machines continuously scan the genome for

DNA mismatches or damage

Most DNA mismatches or damage is only

temporary

• Fixed by DNA repair mechanisms

• All living cells are provided with a plethora of DNA repair mechanism to preserve genomic integrity

The DNA double helix provides

two copies of genetic information, one in each strand

When there is damage or mutation to one strand, the other strand serves as

the source of information to restore the other strand through complementary base pairing

Most damage creates structures that are

not seen in undamaged DNA< so the intact strand is readily distinguished from the damaged strand

The double helix is well suited for

reliably carrying genetic information

Basic mechanism of DNA repair

1. DNA damage/mutations is recognized and segement is excised by nucleases

2. A repair DNA polymerase binds to the 3’ hydroxyl end of the cut DNA strand and fills in the missing nucleotide using the undamaged DNA as a template

3. The nick in the helix is sealed by DNA ligase

Mismatch repair happens

right after DNA is replicated

How do we know which is the correct strand?

There are DNA nicks in the newly synthesized strand (the correct strand) that distinguish it from the parental strand

How does the accuracy of DNA replication change through the stages of proofreading and mismatch repair?

The error rate improves significantly at each step:

• No proofreading: 1 mistake per $10^5$ nucleotides.

• With proofreading: 1 mistake per $10^7$ nucleotides.

• With mismatch repair: 1 mistake per $10^9$ nucleotides.

Compare Depurination and Deamination in terms of what is lost from the DNA strand.

• Depurination: Spontaneous loss of an entire purine base (Guanine or Adenine), leaving an empty sugar-phosphate site.

• Deamination: Loss of an amino group from a base; specifically, Cytosine is converted into Uracil.

Note: Neither process breaks the phosphodiester backbone.

Compare the consequences of unrepaired Deamination vs. Depurination after a round of DNA replication.

• Deamination: Results in a substitution mutation (e.g., C-U leads to a C-G pair becoming an A-T pair).

• Depurination: Results in a deletion mutation because the replication machinery skips the missing nucleotide.

How does UV light specifically damage DNA, and what is the immediate physical consequence for the double helix?

• Damage: It causes Pyrimidine Dimers (specifically Thymine dimers), where UV light triggers the formation of covalent bonds between two adjacent pyrimidine bases on the same strand.

• Consequence: This creates a distortion (kink) in the DNA backbone, which causes DNA polymerase to stall because it cannot "read through" the dimer.

90% of pyrimidine dimers are repaired through

DNA repair mechanisms within minutes

Skin cells exposed to UV light from the sum are especially susceptible to

this type of DNA Damage (pyrimidine dimers)

Basic mechanism of excision repair

1. DNA damage/mutations is recognized and segment is excised by nucleases

2. A repair DNA polymerase binds to the 3’ hydroxyl end of the cut DNA strand and fills in the missing nucleotide using the undamaged DNA as a template

3. The nick in the helix is sealed by DNA ligase

Excision repair systems are active throughout the cell cycle

Double stranded DNA breaks are repaired in one of two ways

Non-homologous end joining or homologous recombination

Nonhomologous end joining (NHEJ)

• The cell tries to quickly repair the break before the two fragments drift apart

• Error prone process (loss of nucleotides at repair site)

Homologous recombination (HR)

• Homologous DNA (sister chromatid) can serve as template for repair

• Can only occur if DNA break occurs shortly after DNA has been replicated

• Error-free process

What is Nonhomologous End Joining (NHEJ), and what is its primary biological "cost"?

It is a "quick and dirty" repair mechanism for double-stranded DNA breaks where the broken ends are polished by nucleases and ligated back together. The cost is the loss of nucleotides at the repair site, potentially leading to a loss of genetic information if the break occurs within a gene.

The double-stranded DNA break-nonhomologous is caused by

mishaps at the replication fork, radiation, and various chemical “assaults”

The cell has no mechanism to replace information lost in a

double stranded break (redundancy of DNA double helix is gone)

How are double-strand DNA breaks repaired by homologous recombination, and when does this process occur?

Homologous recombination repairs double-strand breaks using an identical DNA molecule (sister chromatid) as a template.

Steps:

• Broken DNA ends are processed by nucleases

• The damaged strand invades the homologous, undamaged DNA

• DNA synthesis uses the intact strand as a template

• The break is repaired accurately with no loss of nucleotides

This process occurs only shortly after DNA replication → i.e. befor cell division

How does Homologous Recombination repair a double-strand break without losing genetic information?

• Strand Invasion: A nuclease digests the 5' ends, allowing a broken 3' end to "invade" a nearby undamaged homologous DNA duplex.

• Branch Migration & Synthesis: Repair Polymerase uses the undamaged strand as a template to elongate the invading strand.

• Release and Ligation: The invading strand is released, base-pairs back with its original partner, and DNA Ligase seals the break for an accurate, "invisible" repair.

Polymerase Chain Reaction (PCR) is a technique used in the laboratory to

amplify small segments of DNA — “molecular photocopying”

Significant amounts of DNA are necessary for

many molecular and genetic analyses — would be impossible without PCR

What is PCR used for

• Amplifying gene to study it or the product of it

• Detection of bacteria or viruses — e.g. Covid PCR test

• Studying and mapping genomes (finding the sequence of a genome and identifying genes)

• Diagnosis of genetic disorders

What do we need to replicated DNA?

1. Template sequence

2. A way to separate the double stranded template DNA

   1. Heat takes the place of helicase

3. Primers

   1. We use synthesized DNA primers, so we eliminate the need for primase, nuclease, and ligase

4. Enzymes - DNA polymerase

5. Nucleotides

How are PCR primers designed in relation to the target DNA sequence, and in what direction are they always written?

• Forward Primer: Matches the sequence of the 5' to 3' (top) strand; it binds to the 3' end of the bottom strand.

• Reverse Primer: Is the "Reverse Complement" of the 5' to 3' (top) strand; it binds to the 3' end of the top strand.

• Writing Convention: Primers are single-stranded DNA and are always written in the 5' to 3' direction.

What are the three steps of a PCR cycle, and why is an excess of primers necessary?

• 1. Denaturation: Heat to separate the DNA strands.

• 2. Annealing: Cool to allow primers to bind to the target sequence.

• 3. Extension (Synthesis): DNA polymerase adds dNTPs to the 3' end of the primers.

• Primer Excess: Ensures that the template strands bind to the primers rather than re-annealing to each other.

• We do not have replication bubbles and bidirectional replication in PCR

What is the mathematical relationship between PCR cycles and DNA copies, and when does the desired target-length product first appear?

• Yield: It is an exponential increase ($2^n$). After 25–35 cycles, you have roughly 1 billion copies.

• Target Length: The discrete, double-stranded "target" product (bounded exactly by the primers) first appears in the 3rd cycle.

What are the necessary steps to obtain a high concentration of a specific gene of interest from a sample of whole cells?

• Lyse the cells and isolate the total genomic DNA.

• Use the genomic DNA as a template in a PCR reaction.

• Use primers specific to the gene of interest to selectively amplify that segment into billions of copies (genomic clones).

In Agarose Gel Electrophoresis, how is DNA separated, and in which direction does it move?

• Separation: DNA is separated by size. Shorter fragments travel faster/further, while longer fragments travel slower.

• Direction: DNA moves toward the positive electrode because the DNA backbone is negatively charged.

Plasmids are a

genetic structure in a cell that can replicate independently of the chromosomes

Plasmid structure

small circular double stranded DNA

Plasmids are used frequently in the laboratory manipulation of

genes

Can insert our amilified gene of interest into a

plasmid → many downstream applications like protein production or gene sequencing.

What are the two key enzymatic steps required to insert a DNA fragment into a plasmid?

1. Cleavage: A restriction enzyme cuts the circular plasmid DNA to open it up.

2. Ligation: DNA Ligase creates a covalent linkage between the DNA fragment and the plasmid.

• Result: Recombinant DNA (a single circular molecule containing the original plasmid plus the new insert).

How does Bacterial Chromosomal DNA differ from Plasmid DNA in a laboratory setting?

• Chromosomal DNA: Essential, massive, and carries the "blueprints" for the cell. It is usually not manipulated directly for cloning.

• Plasmid DNA: Non-essential, small, and used as a vector to carry foreign DNA because it replicates independently and is easy to isolate/manipulate.

What is the natural biological function of Restriction Enzymes, and how are they named?

• Function: They are an ancient bacterial defense mechanism used to protect against invading bacteriophages (viruses) by cleaving the viral DNA.

• Naming: They are named after the species of bacteria they were naturally isolated from (e.g., EcoRI comes from E. coli).

Compare the two types of DNA ends produced by different restriction enzymes: Blunt ends vs. Sticky ends.

• Blunt Ends: The enzyme cuts straight through both strands at the same position (e.g., HaeIII).

• Sticky Ends: The enzyme cuts the DNA in a staggered way, leaving short, single-stranded overhangs (e.g., EcoRI and HindIII).

• Note: Sticky ends are highly useful in cloning because they can easily base-pair with complementary sequences.

What is a characteristic feature of the cleavage sites (recognition sequences) for most restriction enzymes?

They are usually palindromic sequences, meaning the 5’ to 3' sequence is the same on both the top and bottom strands (e.g., GAATTC for EcoRI).

In the context of recombinant DNA, why are "sticky ends" alone insufficient to join two DNA fragments permanently?

Sticky ends stay together via hydrogen bonds between complementary bases, but physical gaps remain in the sugar-phosphate backbone. DNA Ligase is required to catalyze the formation of phosphodiester bonds, sealing those gaps and creating a continuous DNA molecule.

How can you ensure a target gene amplified by PCR can be inserted into a specific plasmid vector?

By adding the sequence of a specific restriction site (e.g., EcoRI) to the 5’ ends of your PCR primers. During amplification, these sites are incorporated onto both ends of the target gene, allowing it to be cut by the same restriction enzyme used to open the plasmid.

In the provided diagram, why must both the target gene and the plasmid be treated with the same enzyme (e.g., EcoRI)?

To create complementary sticky ends. For the target gene to successfully ligate into the plasmid, the single-stranded overhangs on the gene must match the base-pairing sequence of the overhangs on the opened plasmid.

What are the two types of chemical bonds formed when inserting a target gene into a plasmid vector?

• Hydrogen Bonds: Formed first between the complementary bases of the sticky ends.

• Phosphodiester Bonds: Formed second by DNA Ligase to seal the sugar-phosphate backbone gaps.

Summarize the complete molecular cloning workflow: PCR → Restriction → Ligation.

• PCR: Amplifies the gene (primers include restriction sites).

• Restriction: Enzymes cut the gene and plasmid to create matching ends.

• Ligation: DNA Ligase joins the gene and plasmid into one continuous circular molecule.

How are specific restriction enzyme sites (e.g., NcoI or NotI) added to the ends of a DNA fragment during PCR for cloning?

• Primer Design: The desired restriction site sequence is added to the 5’ end of both the forward and reverse PCR primers.

• Initial Rounds: In the first round, the primers anneal to the template; the new strand includes the primer's sequence.

• Incorporation: By the second and subsequent rounds, the added restriction site sequence becomes a permanent part of the double-stranded PCR product.

• Final Preparation: The resulting PCR product is then digested with the corresponding restriction enzymes to create the sticky or blunt ends needed for ligation into a plasmid.

What is a GFP fusion protein, and how is it created at the genetic level?

• Definition: A chimeric protein consisting of a target protein joined to Green Fluorescent Protein (GFP).

• Mechanism: The DNA sequence for the target protein is ligated into a plasmid immediately adjacent to the GFP gene, under the control of a single promoter.

• Expression: The cell transcribes a single mRNA and translates a single polypeptide chain where both proteins are physically linked.

What is the primary advantage of using GFP fusion proteins in live-cell imaging?

It allows researchers to monitor the localization and expression levels of a specific protein LIVE over time. Because the tag is genetic, you can watch how proteins move, cluster, or degrade within a living cell without needing to fix or kill the sample.

In a GFP expression vector, what is the role of the promoter located upstream of the Multiple Cloning Site (MCS)?

The promoter acts as the binding site for RNA Polymerase, initiating the transcription of the fused gene sequence. It determines when, where, and how much of the fusion protein the cell will produce.

DNA sequencing determines

the order of the nucleotides that make up the DNA molecule of interest

There are several methods/type of DNA sequencing that are

used for different purposes

Dideoxy (Sanger) method

Older method, which is routinely used for sequencing small pieces of DNA (such as in our plasmid)

What is the structural difference between a dNTP and a ddNTP, and how does this affect DNA synthesis

• dNTP (deoxyribonucleoside triphosphate): Has a 3' -OH group, which is necessary for DNA polymerase to add the next nucleotide.

• ddNTP (dideoxyribonucleoside triphosphate): Lacks the 3' -OH group (it has a 3' -H instead).

• Effect: Incorporation of a ddNTP acts as a chain terminator, meaning no further nucleotides can be added to that DNA strand.

How does Sanger (Dideoxy) Sequencing produce a readable DNA sequence?

1. A DNA template is mixed with a primer, DNA polymerase, regular dNTPs, and small amounts of fluorescently labeled ddNTPs.

2. Synthesis occurs until a ddNTP is randomly incorporated, terminating the chain.

3. This creates a diverse set of DNA fragments of varying lengths, each ending in a labeled base.

4. Fragments are separated by size via capillary electrophoresis, and a laser detects the color of each fragment to generate a chromatogram.

In Sanger sequencing, why do we only sequence one of the two strands of double-stranded DNA?

If both strands were sequenced simultaneously in the same reaction, the fluorescent signals from the two complementary sequences would overlap, making the resulting chromatogram unreadable. Using one primer ensures that only one specific strand is amplified and read.

Sequencing works similar to PCR with

one primer and FL -labeled ddNTPs