Module 1

Nucleotide

sugar, nitrogenous base, and phosphate

make up DNA

Nucleoside

sugar and nitrogenous base

Deoxyribose

the DNA sugar

has a hydrogen on the 2’ carbon

Purines

nitrogenous bases with two rings

includes adenine (A) and guanine (G)

Pyrimidines

nitrogenous bases with one ring

includes cytosine (C), thymine (T), and uracil (U)

5’ End of DNA and RNA

phosphate group

3’ End of DNA and RNA

hydroxyl group

DNA Polymerase

enzyme that synthesizes DNA 5’ to 3’

Phosphodiester Bond

covalent bond linking nucleotides

Hydrogen Bonds

bond type between nitrogenous bases/base pairs formed through partial charges

two between adenine and thymine and three between cytosine and guanine

Ribose

the RNA sugar

has a hydroxyl on the 2’ carbon

Transcription

synthesis of RNA from DNA template

occurs in the nucleus and mitochondria

occurs 5’ to 3’

Genes

units of DNA that are transcribed by RNA polymerase to RNA

Template/Antisense Strand

strand of DNA read from 3’ to 5’ when RNA polymerase synthesizes RNA

Coding/Sense Strand

matches the new RNA strand with thymine instead of uracil

Messenger RNA (mRNA)

RNA that will be translated into protein

Regulatory Sequences

control when and where a gene is expressed

Enhancers & Silencers

cis-regulatory elements that fine tune the spatiotemporal pattern of gene expression

may be distant from the gene they control

Exons

expressed sequences

Introns

intragenic or intervening sequences

pre-mRNA

contains exons and introns

before splicing

Open Reading Frame (ORF)/Coding Sequence (CDS)

within the exons from start codon to stop codon

translated into protein

Promoter

immediately 5’ to transcriptional start site

docking site for RNA polymerase II and other transcriptional proteins

Start Codon

ATG which codes from methionine (met)

Stop Codons

TAA, TAG, and TGA

Untranslated Regions (UTRs)

contained in the first and last exon

5’ Untranslated Region

between transcriptional start site and translational start site

has roles in translation initiation and ribosome binding

3’ Untranslated Region

between stop codon and end of transcript

roles in poly(A) addition, mRNA export, stability, and localization, and translation efficiency

5’ Cap

co-transcriptional pre-mRNA processing in which a 7-methylguanosine is linked with 5’-5’ triphosphate linkage

confers stability and has roles in mRNA splicing, transport, and interaction with ribosomes

Splicing

co-translational processing that generates mature mRNA by getting rid of introns

Splice Donor & Acceptor Sites

sequences recognized by splicing machinery

Branch Site

sequence about 20 nucleotides from splice acceptor site that is recognized by splicing machinery

Spliceosome

consists of small nuclear ribonucleoproteins

Alternative Splicing

mRNA transcripts may not always be spliced the same way

SR Activator Proteins

serine and arginine rich proteins that bind to splicing enhancers to favor exon inclusion

hnRNP Repressor Complexes

bind to splicing silencers to favor exon skipping/exclusion

Poly(A) Signal Sequence

sequence in 3’ UTR required for transcription termination

AAUAAA

single mRNA may have multiple/alternative ones

Poly(A) Tail

posttranslational processing in which poly(A) polymerase adds non-templated adenosines to 3’ end of cleaved mRNA

length varies

aids in nuclear export and translation and stabilized mRNA

Peptide Bonds

hold amino acids together

N-Terminus

amino portion of an amino acid chain

C-Terminus

carboxyl portion of an amino acid chain

Ribosome

made of rRNAs and proteins in two subunits

critical for translation

Transfer RNAs (tRNAs)

bring amino acids to the ribosome

covalently carry amino acids with ester bonds

Anti-Codon

found on tRNAs and base pair with mRNA

Initiator tRNA

binds to 5’ cap and recognizes start codon

needed to begin eukaryotic translation

carries Met

Kozak Consensus Sequence

sequence that surrounds the start codon (AUG) and indicates which AUG is the start codon

Primary Protein Structure

amino acid sequence

Amino Acids with Nonpolar Side Chains

Gly, Ala, Val, Leu, Ile, Met, Phe, Trp, and Pro

Amino Acids with Polar Uncharged Side Chains

Ser, Thr, Tyr, Asn, Gln, and Cys

Amino Acids with Polar Charged Side Chains

Acidic (-): Asp and Glu

Basic (+): Lys, Arg, and His

Secondary Protein Structure

hydrogen bonds within the backbone allow formation of alpha helices and beta sheets

Protein Tertiary Structure

covalent (disulfide) and noncovalent bonds between side chains in a single polypeptide

folding is crucial for function

Protein Quaternary Structure

covalent and noncovalent interactions between polypeptides

Karyogram

a standardized visual profile or photograph of a complete set of an organism’s chromosomes

Pangenome

the complete set of all genetic sequences and variations found across multiple individuals within a single species or population

rRNA Genes

arranged as large tandem repeats

tandem arrays on the p-arms of acrocentric chromosomes

tRNA Genes

numerous and scattered throughout the genome

many in tandem repeats

Retrotransposons

transcribed RNA is converted to DNA by reverse transcriptase, and that DNA integrates into genomic DNA

Long Interspersed Nuclear Element-1 (LINE-1)

retrotransposon with an internal promoter that encodes proteins

ORF2 encodes a protein with endonuclease and reverse transcriptase functions

copies itself and moves around (autonomous)

Short Interspersed Nuclear Element (SINE)

retrotransposon with an internal promoter that does not code for protein

only a few can still make RNA

cannot transpose without help from LINE-1

Satellite DNA

large with tandem repeats found in the centromere

Microsatellite DNA

small with tandem repeats and dispersed

Non-Allelic Homologous Recombination

chromosome misalignment during crossing over

can lead to tandem duplication/deletion

Replication Slippage

expands and contracts microsatellites/short tandem repeats

Polymerase Chain Reaction (PCR)

invaluable method for amplifying DNA that requires two oligonucleotide primers, each complementary to one strand of the target, to prime DNA synthesis by thermostable DNA polymerase

Forward Primer

matches 5’ end of upper strand

anneals to 3’ end of lower strand

Reverse Primer

matches 5’ end of lower strand

anneals to 3’ end of upper strand

Real-Time PCR (qPCR)

quantitative and relies on production and measurement of fluorescence throughout the procedure

measures amount of product at the end of each cycle

Competitive Allele Specific Taqman PCR

version of real-time PCR that can detect rare variants with a primer that binds to the mutant allele

uses a probe that binds to the product with fluorescence and a quencher

the Taq polymerase chews up the probe, separating the fluorescence and the quencher, allowing it to glow

Droplet Digital PCR

ultrasensitive counting methodology for PCR using fluorescent droplets and the Taqman assay

Array Comparative Genome Hybridization (a-CGH)

used for copy number variant detection and quantification

uses a spot library of know gDNA that cover the genome or a portion of the genome to solid surface and fluorescence

a red product indicates a duplication and a green product indicates a deletion

Fluorescence In Situ Hybridization (FISH)

used for chromosomal localization

fluorescent labeled probe is specific to DNA region of interest

RT-PCR

detects and quantifies mRNA after it has been converted to cDNA by reverse transcriptase

Nanostring nCounter

used to quantify mRNA using a hybridization method that directly counts RNA molecules

RNA In Situ Hybridization (RNA ISH)

used to visualize RNA

Western Blot

used to detect and quantify protein by using a gel then transferring to a membrane

Immunostaining

used for protein localization

Chromatin

DNA and proteins (DNA wrapped around histones, forming nucleosomes)

folds into fibers that loop, compress, and coil, allowing DNA compaction

AT rich areas are more condensed, and GC rich areas are less condensed

Interphase

cell cycle phase including G1, S, and G2

phase when chromosomes are relatively open and most gene expression happens

Euchromatin

open chromatin

generally gene-rich

required for gene expression

Heterochromatin

condensed chromatin

generally gene-poor

CpG Dinucleotide

C next to G

often underrepresented in the genome since the C gets changed to a T after methylation because 5mC is unstable

CpG Islands

long tracts of unmethylated CpG dinucleotides

overrepresented in promoters and often mark active promoters when unmethylated

are evolutionarily protected

methylation turns gene off

DNA Methyltransferase

adds methyl to DNA

TET

oxidizes 5mC as first step to base removal and repair

Enhancers

DNA sequences that may build complexes that increase transcription

may be many places in relation to gene and can be far away

orientation does not matter

found in nucleosome depleted regions

Silencers

DNA sequences that may build complexes that repress transcription

may be many places in relation to gene and can be far away

orientation does not matter

found in nucleosome-depleted regions

DNA Binding Domains (DBD)

where transcription factors bind enhancers

DNA sequences/motifs recognized by specific transcription factors

Mediator Complex

bridges transcription factor activation domains and basal transcription machinery for long-range interactions

Topologically-Associating Domains (TAD)

where promoter-enhancer interactions occur

flanked by CTCF binding sites that mark where the ring that travels down chromatin to create loops stop

Master Regulators

may coordinately control expression of many cell type specific genes

Histone Acetylation

loosens DNA/histone association, favoring open chromatin, by making the histone more neutrally charged

associated with active gene expression

balanced by HAT and HDAC

Histone Methylation

can be associated with gene expression activation or repression, depending

does not cause a charge change on histone

balanced by HMT and HDM

RNA-seq

quantifies global transcript populations

DNAse-seq

used DNAse I hypersensitive sites to define open chromatin sites

FAIRE-seq

uses formaldehyde crosslinking to define chromatin open sites

ATAC-seq

uses transposase-accessible chromatin to define open sites

DNA is cut where it is open and adapters used for sequencing

advantages: less sample input and faster experiment

ChIP-seq

identifies global protein/DNA interactions that signify active regulatory regions through immunoprecipitation

CUT&RUN

more sensitive version of ChIP-seq

Chromosome Conformation Capture (3C)

assigns enhancer to a gene

has derivatives