LC 4

Homologous Recombination (HR)

(homology-dependent repair') DNA exchanges or uses matching DNA sequences to repair damaged DNA or create new combinations of genes. ACCURATE

non-homologous end joining (NHEJ)

NHEJ repairs double-strand DNA breaks by directly joining the broken ends without using a homologous DNA template. SIMPLE

RecBCD complex polypeptides

RecB, RecC, and RecD

RecBCD complex

unwinds and processes broken DNA ends, creating a single strand that can pair with matching DNA for repair.

Chi site

DNA sequence that helps RecBCD prepare broken DNA for repair by RecA (GCTGGTGG)

RecA

finds matching DNA and helps the broken strand invade it so the DNA can be repaired.

RuvC complex

Cuts the DNA junction so the strands can separate and be rejoined.

RuvA protein

Recognizes and binds to Holliday junction.

RuvB complex

Uses energy (ATP) to move the DNA strands through the junction.

How do researchers design linear segments of engineered DNA that can be sent into living cells for integration into specific genomic locations?

Researchers add matching homology arms to engineered DNA so homologous recombination can insert it at a specific location in the genome.

site-specific recombination (SSR)

Recombines DNA at specific, short recognition sequences. RAPID

Cre recombinase

An enzyme that cuts and rejoins DNA at specific sites.

loxP recognition sites

Short DNA sequences that Cre recognizes to recombines the DNA between or around them

non-homologous end joining repair

repairs a double-strand DNA break by joining the broken ends together WITHOUT using a matching DNA template.

Ku70/Ku80

hold broken DNA ends together

Artemis

trims damaged DNA

Ligase IV

seals ends back together.

antibody diversity

V(D)J recombination + Somatic hypermutation

V(D)J recombination

Random DNA segment joining

Somatic hypermutation

Base deamination

Restriction endonuclease (REase)

cuts unprotected foreign DNA

DNA methyltransferase (MTase):

Adds methyl groups to the bacterium's own DNA to mark and protect it.

Methyl groups

protect DNA

What happens in REase and MTase are disturbed?

MTase fails= REase might cut the bacterium's own DNA.

REase fails= foreign DNA survies and enters cell

CRISPR array

short, repeated DNA sequences separated (repeats) by unique DNA sequences (spacers)

spacers origin

DNA of viruses or other foreign genetic material.

cas genes

code for bacteria fighting protein

Streptococcus thermophilus 2007 paper

Phage exposure causes bacteria to acquire new spacers that match the corresponding phage genome

spacer acquisition (immune memory)

Cas 1 + Cas 2 insert spacers

interference process (immune response)

Cas 9 cuts foreign DNA

cRNAs

Come from CRISPR array. Guides Cas proteins to matching foreign DNA

protospacer-adjacent motif (PAM)

allows Cas9 to recognize and bind the target DNA so it can cut the foreign DNA

How is sRNA used?

to edit specific genes

forward genetic analysis

Starts with an interesting phenotype (observable trait) and looks for the gene responsible. Phenotype → Gene

Reverse genetic analysis

Starts with a gene of interest and studies what happens when the gene is changed to determine its function. Gene → Phenotype

Cry5B/C. elegans central question

how does Cry5B kill C. elegans? Used Bt toxins

Bre paper subject

If a bre mutant is crossed with a wild-type mate, the babies will have wild-type phenotypes because they inherit a normal copy of the gene from the wild-type parent.

Recessive

a mutant trait only appears when an organism has two copies of the recessive mutant allele.

gain-of-function

new protein functions that didn't exist before

loss-of-function

mutations decrease or abolish protein function (bre mutation)

complementation/rescue experiment

Introduces a normal copy of a gene into a mutant organism to see if it restores normal function.

Cry5B toxins

works by binding to specific sugar molecules on the surface of intestinal cells.

glycolipid extraction experiments bre results

showed that Cry5B toxin likely binds to specific glycolipids

How do you investigte traits?

Identify mutants, compare genes, introduce a normal copy to identify which type of mutation it is

Random transposon insertion + PCR screening

Transposons randomly insert into DNA and disrupt genes. PCR is used to find organisms where the transposon inserted into the gene of interest.

Homologous recombination

Uses matching DNA sequences to replace or disrupt a specific gene with a modified DNA sequence.

RNA interference (RNAi)

Uses small RNA molecules to target and break down a gene's mRNA, reducing protein production without changing the DNA.

CRISPR-Cas9

Uses a guide RNA to direct Cas9 to a specific DNA sequence. Cas9 cuts the DNA, and repair can create mutations that disrupt the gene.

Ways to disrupt a gene (4)

random transposon insertion followed by PCR screening, homologous recombination, RNA interference (RNAi), and CRISPR-Cas9

How does repair of a Cas9-induced double-strand break by NHEJ disrupt gene function?

Makes small insertions or deletions. These can change the reading frame or create a premature stop codon, preventing the gene from making a functional protein

SHP1/2

two genes in Arabidopsis thaliana (a model plant) that have similar, overlapping functions.

Knocking out both genes

causes a strong abnormal phenotype, such as changes in shoot branching.

Knocking out either gene

did not cause the same phenotype because the other gene could compensate for the missing gene.

Left homology region

Matches the DNA just before the target gene

Right homology region

Matches the DNA just after the target gene

Explain how homologous recombination can be used to replace a yeast gene with an antibiotic-resistance marker

How would you design PCR primers to amplify a selectable marker while adding homology regions corresponding to the ends of a target gene?

Design primers that bind to the selectable marker. Add extra sequences to the 5′ ends of the primers that match the DNA just before and just after the target gene.

How can antibiotic resistance make it easier to isolate successful gene knockouts?

Antibiotic resistance acts as a selectable marker.

plasmids

They are small, circular DNA molecules that can replicate inside cells and carry genes researchers want to study or manipulate.

Basic elements of research plasmid

origin of replication (ori), selectable marker, and multiple cloning site (MCS)

origin replication (ori)

Allows the plasmid to copy itself inside a cell

Selectable marker

Allows researchers to identify cells containing the plasmid, often through antibiotic resistance.

multiple cloning site (MCS)

A region containing restriction enzyme cut sites where researchers can insert DNA of interest.

plasmid pBR322

Good for research because of its’ compact size and versatility.

Molecular cloning steps

purifying vector and insert, restriction digestion, DNA ligation, transformation into E. coli, and selection of colonies.

purifying vector and insert

Isolate the plasmid (vector) and the DNA fragment (insert) you want to clone.

restriction digestion

Use restriction enzymes to cut the vector and insert at specific DNA sequences, creating compatible ends.

DNA ligation

Use DNA ligase to join the insert and vector together.

transformation into E.coli

Introduce the recombinant plasmid into bacterial cells.

selection of colonies

Grow the bacteria on antibiotic-containing media. Surviving colonies likely contain the plasmid, which can then be checked to confirm the correct insert is present.

clone

a bacterial colony containing copies of the same recombinant plasmid with the DNA insert of interest.

vector

plasmid in which you wish to insert a new gene

insert

ene of interest; this sample is often a PCR product

two main methods for getting plasmid DNA into E. coli cells

Electroporation and Heat Shock

How could an expression plasmid be used to produce a human protein such as tyrosinase (TYR) in E. coli?

An expression plasmid can carry the human TYR gene into E. coli so the bacteria can produce tyrosinas

promoter

Starts transcription of the TYR gene into mRNA.

Translation initiation region

Helps the ribosome start translating the mRNA into a protein

His₆ affinity tag

Adds six histidine amino acids to the protein, allowing researchers to easily purify it from other proteins.

Why is amplifying the human TYR CDS directly from genomic DNA problematic for expression in E. coli?

Human genomic DNA contains introns, which E. coli cannot remove because bacteria lack the machinery for eukaryotic RNA splicing.

How can reverse transcriptase and RNA-derived complementary DNA (cDNA) overcome the DNA E. coli problem?

Use reverse transcriptase to make cDNA from mature human TYR mRNA. The cDNA contains the coding sequence without introns, allowing E. coli to express the gene more effectively.

5′ overhangs

Single-stranded DNA extensions at the 5′ ends. (stiky ends)

3′ overhangs

Single-stranded DNA extensions at the 3′ ends.

blunt ends

Both DNA strands are cut at the same position, leaving no overhangs.

Left homology region

Matches the DNA just before the target gene

Right homology region

Matches the DNA just after the target gene

Explain how homologous recombination can be used to replace a yeast gene with an antibiotic-resistance marker

inserting a DNA sequence containing the marker into the gene's location.

Premier plant model

arabidopsis

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeat