Genetics exam 5

method for detecting proteins in developing animals

immunolocalization

method for detecting mRNA in developing animals

in-situ hybridization

toolkit genes

genes that determine the body plan and the number, identity, and pattern of body parts

homeotic transformation

one body part is replaced by another

bithorax homeotic transformation in flies

2 body segments develop wings instead of 1

antennapedia homeotic transformation in flies

a pair of legs develop instead of antenna

Hox genes

genes that regulate the identity of body parts

method of expression for Hox genes

expressed in spatially restricted domains

Hox proteins

transcription factors encoded by Hox genes

homeodomain 

a sequence in common of Hox proteins that act as a DNA-binding domain

purpose of comparing protein sequences by sequence alignment

identify highly conserved regions and amino acids with similar functions

primary role of Hox genes during normal Drosophila development

determine the identity of segments

maternal effect genes

genes carried by the female that are required for early embryonic development

bicoid

a maternal effect gene that sets up the anteroposterior axis of the embryo

bicoid mutant

missing the anterior region (headless)

classes of zygotic genes

GAP genes, pair rule genes, segment polarity genes

GAP genes

define contiguous blocks of segments

pair rule genes

define a pair of segments

segment polarity genes

affect patterning in each segment

phenotype of mother (with mutant offspring) in a mitochondrial pedigree

affected 

phenotype of mother (with mutant offspring) in a maternal effect gene pedigree

unaffected

sex-lethal protein

affects alternative splicing when expressed - leads to female development

female-specific splicing

sex-lethal protein expressed → transformer expressed → alternative splicing → female doublesex protein molecule activates female genes

default splicing

stop codon prevents sex-lethal protein from being synthesized → stop codon leads to transformer NOT expressed → male doublesex protein represses female genes 

mRNA involved in sex determination for Drosophila

sex-lethal, transformer, doublesex

method used by mRNA-binding proteins to determine cell lineages

repression of mRNA translation

controls developmental timing in C. elegans and other species 

microRNA; let-7-lin-41 interaction

wild-type C. elegans development time

4 stages to reach adulthood

let-7 loss of function (C. elegans development)

late transition to adulthood

lin-41 loss of function and let-7 overexpression (C. elegans development)

early transition to adulthood

genotype of IA (bcd gene)

bcd/bcd- (heterozygous)

genotype of IB (bcd gene)

bcd/bcd- OR bcd-/bcd- (heterozygous or homozygous recessive)

genotype of IIB (bcd gene)

bcd-/bcd- (homozygous recessive)

genotype of IIC (bcd gene)

could be homozygous recessive, homozygous dominant, or heterozygous

southern blot

probe = DNA fragments, in vitro

northern blot

probe = RNA fragments, in vitro

western blot

probe = antibodies/proteins, in vitro

PCR and RT-PCR

probe = DNA primers, in vitro

FISH

probe = DNA fragment, in vivo

in situ hybridization

probe = RNA fragment, in vivo

immunofluorescence

probe = antibodies/proteins, in vivo

agarose gel electrophoresis

used to separate DNA/RNA fragments and proteins based on size and shape

blotting method

sample is separated → molecules are blotted and transferred via capillary action → membrane is hybridized with probe and incubated → autoradiogram used to visualize

main reactions of PCR

denature → anneal → extend

purpose of PCR

amplifying and detecting DNA

reverse transcriptase

uses RNA as a template to synthesize a complementary DNA strand in RT-PCR

two components of reverse-transcription PCR (RT-PCR)

reverse transcriptase and cDNA (complementary DNA)

quantitative real-time PCR (RT-qPCR)

measures the amount of DNA product in “real time” during each PCR cycle

concentration difference between two samples of RT-qPCR

2 to the power of the cycle difference

cycle threshold (C_T)

used to measure the amount of DNA in each sample

key steps in generating recombinant DNA molecules

select correct vector → restriction enzyme digestion → ligation of donor DNA and vector DNA → transformation → confirmation

methods to detect which recombinant cells now have plasmid DNA

antibiotic resistance, perform PCR and run a gel, screen for expression of a reporter gene

plasmid vectors

small, easy to work with, high copy number, accept inserts of small sizes (up to 5 kb)

phage vectors

accept inserts up to 15 kb, more steps required to construct the clone, require packaging DNA into the phage (more time-consuming)

fosmid vectors / BAC

accepts inserts of large sizes, less clones are required

essential features of cloning vectors

cloning site, selection marker gene, origin of replication

genomic library

a collection of clones that cover the entire genome of an organism (uses vectors with large insert size)

cDNA library

a collection of clones that represent mRNAs present in the sample (uses plasmid vectors)

structure of 2’,3’-dideoxynucleotides

3’ position does not have a hydroxyl group and cannot form a phosphodiester bond with the next dNTP

how to read a dideoxy sequencing gel

start with the smallest fragment, determine the base pair, and continue up to the largest fragment to determine the sequence of the template strand

reagent that results in chain termination in Sanger sequencing

dideoxynucleotides

strategies for creating site-specific double-stranded breaks

zinc-finger nucleases, TALENs, CRISPR

zinc-finger nucleases

creates a DNA binding domain that only targets specific nucleotides

TALENs (transcription activator-like effector nucleases)

engineers a nuclease that carries a site-specific protein

CRISPR genome engineering

used to selectively change the sequence of the genomic DNA = single guide RNA + Cas9 enzyme

CRISPR-Cas9

a DNA endonuclease from bacteria that functions with CRISPR sequences to provide immunity against pathogens

single-guide RNA (sgRNA)

a small RNA molecule that guides the Cas9 endonuclease to make a double-stranded cut in CRISPR

purpose of donor DNA in CRISPR

helps to repair the double-stranded break

overall purpose of CRISPR-Cas9

helps to restore normal function of a mutated gene, which is useful for treating genetic diseases

reverse genetics

begins with cloned DNA or protein and uses it to introduce mutations back into the genome to investigate its function (genotype → phenotype)

bioinformatics

analysis of the info content of entire genomes

comparative genomics

considers the genomes of closely and distantly related species for evolutionary insight

functional genomics

uses a combination of different approaches to understand the functions of many genes

whole-genome shotgun (WGS)

obtaining the sequence of a genome by assembling and sequencing many short segments of genomic DNA (sequence first, map later)

order-clone sequencing

map first, sequence later

Sanger sequencing

more accurate, takes longer/costs more, longer sequence read, easier to assemble

pyrosequencing

less accurate, more cost-effective, no library needed, shorter sequence read, best for genomes with reference sequences available

techniques used in traditional whole genome shotgun (WGS)

genomic library construction, DNA cloning, Sanger dideoxy DNA sequencing

next-generation WGS does not require this technique

prior construction of genomic libraries in microbial hosts

open reading frame

a stretch of nucleotide sequence that is not interrupted by a stop codon in a given translation reading frame

expressed sequence tag (EST)

partial sequence of cDNA that is used to identify transcript ends

reasons why the human genome has more protein products than protein-coding genes

alternative splicing, post-translational modification

RNA-sequencing (RNA-seq)

a genomic method used to identify all of the genes that are transcribed

two-hybrid test

a method used to detect protein-protein interactions, typically performed in yeast

chromatin immunoprecipitation assay (ChIP)

isolates the DNA and its associated proteins in a specific region of chromatin so that both can be analyzed together

steps of ChIP

cross-link proteins to DNA → break chromatin into small pieces → add antibody to target protein and purify → reverse cross-links to separate DNA and protein → amplify and sequence

methods used in reverse genetics

random mutagenesis, targeted mutagenesis, phenocopying (RNAi pathway)