Genetics Unit 4 Exam

Restriction enzymes function

Cut (“digest”) DNA at specific palindromic sequences (“restriction sites”)

palindromic

top strand read 5’ to 3’ is the same as the bottom strand read 5’ to 3’

organismal clones

genetically identical (multicellular) organisms - Dolly the Sheep

Cellular clones

genetically-identical cells (a bacterial colony)

molecular clones

identical molecules (e.g. DNA)

cloning vectors

DNA molecules designed for molecular cloning - 3 key features: polylinker, selectable markers (ampR), and origin of replication

polylinker function

multiple cloning site - a section of DNA containing lots of restriction sites for scientists to choose from to insert DNA fragments

selectable markers

EXAMPLE: antibiotic resistance gene (ampR), allows for selection of bacteria transformed wtih the plasmid

function of the origin of replication

so that the transformed bacteria can replicate (clone) the DNA

plasmid

small, circular DNA molecules that can be used as cloning vectors

how are recombinant plasmids formed?

genomic fragments + plasmids with compatible sticky ends are ligated together with ligase

how is a genomic DNA library created

recombinant plasmids are introduced to bacteria; each bacteria has a different section of DNA incorporated in the genome

why is antibiotic selection performed?

this process eliminates untransformed bacteria, so only the rare bacteria we want survive (those with ampR resistance)

genomic library

collection of cloned genomic fragments transformed into cells

ddNTPS

dideoxyribonucleic acids (no OH groups AT ALL) - no way to form a phosphodiester bond, therefore synthesis immediately stops when one is added

contig

sequence determined by a single sequencing reaction. overlapping sequencing reads can be aligned to assemble larger contigs

a reference genome is NOT

the genome of a single individual, synonymous with “wild type” or “normal,” and 100% perfectly assembled or complete

annotating a genome involves finding and labeling functional elements such as…

genes, promoters, enhancers, etc

what. is cDNA

“complementary DNA” - only contains sequences found in mature mRNAs made by that cell (will not contain promoters and enhancers)

SNPs

“snips” - single nucleotide polymorphisms, they are single-nucleotide differences between genomes

linked SNP

no effect on protein production or function

non-coding, causative SNP 

changes amount of protein produced

coding SNP

changes amino acid sequence

SNP inheritance

SNPs will initially be found in only one compbintation with all the other SNPs on the same chromosome. SNPs on a chromosome will be inherited as a unit unless they are separated by recombination

• you share more SNP alleles and longer DNA segments with closer relatives

haplotype

a group of closely linked genetic markers, or variations like SNPs, on a chromosome that are inherited together from a single parent. people with the same haplotype share all the same completely linked SNPs in a particular region

linked markers

DNPs that do not cause a mutant phenotype, but may still be useful in predicting phenoypes

SNP analysis

Relies on sequencing - an SNP does not change with size of DNA, so PCR + electrophoresis will NOT distinguish between SNPs. SSRs, indels, and copy number variants DO change the number of bases in a region

homologous recombination

DNA exchange between two distinct DNA molecules that have mostly the same sequence

• Can replace a genomic sequence in with the sequence from a donor DNA construct

• If the goal is to knock out the endogenous gene, we can introduce a version of the allele that has an insertion in the middle that renders the allele nonfunctional

why does the homology template contain a copy of the targeted gene disrupted by a neomycin resistance gene?

1. Knocks out the target gene

2. Enables drug selection, so only successfully-edited cells survive
   - Edited stem cells are injected into a blastocyst, which will develop in the womb of a surrogate

mosaic (chimeric) pups

some cells descend from the original blastocyst, but other cells descend from the edited stem cells

knock-in

replacing an allele (the goal is not to simply disrupt the endogenous allele, but to replace with a new one)

conditional knockout

generate mice with alleles that will only be disrupted in some cells, but remain functional in others

NHEJ - non-homologous end joining

• inducing a double-stranded break at a precise location can lead to random mutations if NHEJ is used

• random indels can be introduced at the targeted site

• this technique is often used to generate loss-of-function mutations

• TARGETED MUTAGENESIS

homology-directed repair

• inducing a double-stranded break at a precise location can stimulate homology-directted repair

• based on a homologous repair template

• normally relies on sister chromatids, but if a repair template with homology arms (L and R) is provided, the cell may use that instead

sgRNA

directs Cas9 to cut at specific locations based on base pairing between the spacer region and the target sequence

• Cas9 is the enzyme used to induce double-stranded breaks

two conditions must be met for Cas9 to recognize and cut DNA:

1. The single guide RNA base pairs to the DNA

2. The DNA in the non-complementary strand contains a PAM sequence in the correct location - PAM Is 5’ NGG 3’

expression vector

designed to express a transgene

• must have all the features of a cloning vector: origin of replication, selectable marker (so we can select for transformed bacteria), and multiple cloning sites (so we can insert DNA - a transgene)

role of lac regulatory elements in bacterial expression

many bacterial expression vectors have the polylinker located inside the lacZ gene

• an inserted sequence will be transcribed under the control of lac regulatory elements

• the transcript produced will retain key features of the lacZ 5’ UTR (Shine Delgarno)

• Insertion of a transgene prevents expression of functional lacZ from the plasmid (enables blue-white screening)

X-gal

B-gal activity can be visualized using X-gal

• if B-gal is functional, X-gal is converted into a BLUE chemical

Agrobacterium tumefaciens

can insert pieces of its Ti plasmid into plate genomes

• transform Agrobacterium with plasmids and spray transformed bacteria on plants

hemoglobin

a tetramer of four protein subunits (two of each kind)

• each subunit is encoded by its own gene

fetal hemoglobin composition

2 gamma subunits, 2 alpha subunits (O2 hungry!)

adult hemoglobin composition

2 alpha globin, 2 beta globin

sickle cell disease

caused by a specific missense mutation that changes glutamate to valine

B-thalassemia

any null or amorphic mutation that reduces the amount of B-globin produced

allogenic

from a donor

autologous

from self

Casgevy function for B-thalassemia

• Casgevy introduces mutations in an enhancer of BCL11A

• BCL11A is a transcription factor that promotes the switch from expressing gamma globin (fetal) to B-globin (adult)

• TARGETED MUTAGENESIS