Molecular Genetics

Question	Answer
Nonsense mutation	Substitution of one nucleotide for another. Leads to stop codon - loss of function or expression due to degradation of mRNA. Non-synonymous.
Splice Site Mutation	Substitution of one nucleotide for another. Leads to aberrant splicing. Exon skipping or intron retention. Non-synonymous.
Missense Mutation.	Replacement of single nucleotide by another. Leads to altered amino acid. May affect protein function or stability. Non-synonymous.
Genome-Wide Association Studies	Compares variants across genome in case-control study, replicating SNPs and common diseases. "Hypothesis-free" P value threshold 5 x 10^-8. Associations identify only a small fraction of heritability of each disease studied.
Linkage Disequilibrium	Fine mapping to contstruct haplotypes by genotyping SNPs within identified chromosome region that appears to confer susceptibility. Candidate genes within the region are then sequenced to find DNA variants that can be tested for association with the disease.
Purines	Adenine. Guanine.
Pyrimidines	Cytosine. Thymine. Uracil.
Nonsense-Mediated Decay	Removes Truncated proteins synthesized from mRNAs that have a premature stop codon upstream of 50-55 nucleotides from the last exon-exon junction.
5' cap	5' end of mRNA has a guanine added via a 5' to 5' triphophate linkage, n7 position of guanine is methylated. Facilitates transport of mRNA to the cytoplasm and attachment to the ribosomes. Protects RNA transcript from degradation by endogenous cellular exonucleases.
Polyadenylation	~200 adenylate residues are added to mRNA. Facilitates nuclear export and translation.
mRNA splicing	Non-coding introns in the precursor mRNA are excised, and the non-contiguous coding eons are placed together to form a shorter mature mRNA. Boundary between introns and exons have a 5' donor GT and 3' acceptor AG.
TATA box	25 bp upstream of transcription start site. Involved in initiation of transcription at a basal constitutive level. Mutations can lead to alteration of the transcription start site.
GC box	80 bp upstream of transcription start site. Increase the basal level of transcriptional activity of the TATA box.
Antisense Oligonucleotides	Sequence-specific binding of an antisense oligonucleotide to a target mRNA. Results in inhibition of gene expression at the protein level. Can be used to block an exon-splicing enhancer sequence variant.
Small antisense RNAs	dsRNAs are processed by Dicer to make small 21-23 nucleotide RNAs. Post-transcriptional regulators of gene expression. Regulate cell growth, development and differentiation.
Kozak Sequence	Initiation codon recognition for mRNA translation. AUG encodes methionine and initiates translation within sequence.
Homolog	Genes that share the same sequence.
Ortholog	"Same gene" in different species.
Paralog	similar "duplicated" gene in same species
Pseudogenes	Closely resemble structural genes, but are not functionally expressed. Arise in two ways: 1. duplication events that are rendered silent through acquisition of mutations in coding or regulatory elements. 2. Insertion of complementary DNA sequences produced by reverse transcriptase on a naturally occurring mRNA transcript that lack promotor sequences for expression.
Satellite DNA	Accounts for 10-15% repetitive DNA sequences of human genome. Tandem repeated DNA sequences that are transcriptionally inactive. Clustered around centromeres of certain chromosomes.
Telomeric DNA	Minisatellite DNA. Terminal portion of telomeres of chromosomes. 10-15 kb tandem repeats of 6 bp DNA sequence added to chromosome end by telomerase.
Hypervariable minisatellite DNA	highly polymorphic DNA sequences consisting of short tandem repeats of a common core sequence.
Microsatellite DNA	Tandem single, di-, tri-, and tetra- nucleotide repeat bp sequences located throughout the genome. Variation in repeat number arises by slipped strand misfiring. Used for forensic and paternity tests. Multi-allelic and highly informative.
Short Interspersed Nuclear Elements (SINEs)	5-13% human genome. Repetitive DNA sequences ~300 bp. Called Alu repeats because they contain an AluI restriction enzyme recognition site. Common in regions of GC-rich content.
Long Interspersed Nuclear Elements (LINEs)	20% of human genome. Repetitive DNA sequence interspersed throughout human genome. Most common LINE-1 or L1 element, DNA sequence up to 6000 bp encoding a reverse transcriptase. Common in regions of AT-rich content.
Transposons	Unstable DNA sequences that move spontaneously throughout the genome from one chromosome to another. Alu repeats are flanked by short direct repeat sequences and resemble transposons - could promote unequal recombination, which could lead to mutation in inherited human disease.
DNA methylation	Methylated carbon at 5th position of cytosine pyrimidine ring in CpG dinucleotide. Methylated DNA recruits specific methyl-CpG-binding protein that recruits chromatin-modifying enzyme that silence transcription. 5-methylcytosine is stable epigenetic mark and will be transmitted with cell division.
Histone modification	Loss of H3 and H4 acetylation leads to transcription repression.
Polymorphism	The existence of two or more alleles of at least 1% frequency. 30% structural gene loci are polymorphic. Each individual heterozygous at between 10-20% of all loci.
Transition	Mutation of Purine to Purine (A, G), or Pyrimidine to Pyrimidine (C, T) C to T transitions are the most common mutation due to high frequency of deamination of methylated cytosines.
Transversion	Mutation of Pyrimidine (C,T) to Purine (A,G), or Purine to Pyrimidine
Loss-of-function	Complete loss of the gene product (null allele). Reduced activity or of decreased stability of the gene product (hydromorph).
Haploinsufficiency	Loss-of-function mutations in heterozygous state in which half normal levels of gene product result in phenotypic effects. 50% reduction in protein product causes disease.
Dominant negative	Mutant gene in heterozygous state results in loss of protein activity or function, the mutation gene product interfere with the function of the normal gene product in the corresponding allele. Common in protein dimers and multimers.
Gain-of-function Mutation	Results in increased level of gene expression or the development of a new function of the gene product.
Mendel's 1st law of segregation	Two parental alleles in a diploid individual segregates randomly in gametes.
Mendel's 2nd law of independent assortment	Alleles at different loci distribute randomly into gametes.
De novo variant	Rare: 1-4 per 10^8 bp. (~1 per exome, ~100 per genome) To establish causality for disease: mutations in the same gene in unrelated individuals + functional evidence and clinical correlation
Recombination fraction (Greek letter theta)	Measure of how close two loci are to each other on a chromosome. Measured as proportion, not percentage. 0 = No recombination at all. (completely linked) 0.5 = independent assortment. (unlinked)
Centimorgans (cM)	Unit of measurement for genetic linkage. If 2 loci are 1 cM apart, a cross over occurs once every 100 meiosis (i.e. theta = 0.01). 1 cM ~ 1 million bases, BUT the relationship between linkage map units and physical length is not linear because of recombination 'hotspots'. Male Genetic Map: 2700 cM Female Genetic Map: 4400 cM
Likelihood odds ratio	Probability of data at given linkage value (1-theta)^n(theta)^r ________________________________________ Probability of data with no linkage (0.5)^(n+r) n= number non-recombinants r= number recombinants
Logarithm of the odds (LOD)	LOD (theta) = Log10 (likelihood odds ratio) = Log10 [(1-theta)^n(theta)^r]/(0.5)^(n+r) Estimate theta = r/(n+r) for max LOD. if causal, theta=0, r=0, LOD=n*log10 2 = 0.3n. LOD of -2, proof of non linkage. LOD of +3, proof of linkage.
Relative Risk	pos genotype affected / total pos genotype ______________________________________ neg genotype affected / total neg genotype Larger RR, the stronger the disease association.
Odds ratio	Pos genotype case * Neg genotype control __________________________________________ Pos genotype control * Neg genotype case The larger the OR the higher the risk, the stronger disease association.
Restriction Fragment Length Polymorphism	If a base substitution creates or abolishes the recognition site of a restriction enzyme, it is possible to test for the mutation by digesting a PCR product with the appropriate enzyme and separating the products by electrophoresis.
Amplification-Refractory Mutation System PCR (ARMS PCR)	Allele-specific PCR uses primers specific for the normal and mutant sequences. The most common design is a 2-tube assay with normal and mutant primers in separate reactions together with control primers to ensure the PCR worked.
Oligonucleotide Ligation Assay	A pair of oligonucleotides is designed to anneal to adjacent sequences within a PCR product. If a pair is perfectly hybridized, they can be joined by DNA ligase. Oligonucleotides complementary to the normal and mutant sequences are differentially labeled and the products identified by computer software.
Sanger Sequencing	'Gold standard' Dideoxy chain termination method for mutation detection. The dideoxynucleotides labeled with fluoresent dye lack a hydroxyl group at the 3' carbon position, resulting in a mixture of DNA fragments of different lengths that are separated by capillary electrophoresis resulting in a ladder of DNA sequences of differing lengths. One exon at a time. No separation of molecules. Mixture of reads with and without variant (double peak indicates variant).
Pyrosequencing	Nucleotides are added and removed one at a time with chemiluminescent signals produced after the addition of each nucleotide. Generates quantitative sequence data rapidly.
Next-Generation 'Clonal' Sequencing	In vitro cloning step to amplify individual DNA molecules by emulsion of bridge PCR. Cloned DNA molecules are sequenced in parallel by pyrosequencing or ligation approach. Limitations: high homology regions, triplet repeat expansions, copy number alterations.
Multiplex ligation-dependent probe amplification (MLPA)	High resolution method to detect deletions and duplications. Each probe consists of 2 fluorescently labeled oligonucleotides that can hybridize adjacent to each other to a target gene sequence. When hybridized, the 2 oligonucleotides are jointed by a ligase and the probe is amplified by PCR. Up to 40 probes can be amplified in a single reaction.
mtDNA	37 genes 2 types of ribosomal RNA 22 transfer RNA 13 protein subunits for enzymes (oxidative phosphorylation pathways)
Ribosomes	Site of protein synthesis. 2 different sized subunits with 4 types of ribosomal RNA (rRNA) and ribosomal specific proteins.
Template strand (antisense strand)	One DNA strand of double helix that is used to transcribe mRNA. The transcribed mRNA is a complementary strand (sense strand).
transfer RNA (tRNA)	Amionacyl tRNA synthetase reacts with ATP to covalently bind amino acids to specific anticodon sequence on tRNA molecule to allow AA to be incorporated into polypeptide chain by enzyme peptidyl transferase.
Transcription Factors	Proteins with DNA-binding activity to short nucleotide sequences, usually through helical protein motifs. Transcriptional activation domain and a DNA-binding domain (helix-turn-helix, zinc fingers, leucine sipper, or helix-loop-helix). Trans-acting.
mRNA splicing	Excising non-coding introns in the pre mRNA requires: 5' donor GT, 3' acceptor AG Surrounding short splicing consensus sequences Intronic sequence (branch site) small nuclear RNA (nRNA) molecules and protein
Small interfering RNA (siRNA)	Post-translational gene expression regulator. Short dsRNAs bind to mRNA in sequence-specific manner and result in degradation via a ribonuclease-containing RNA-induced silencing complex (RISC).
MicroRNA (miRNA)	Post-transciptional gene expression regulator. Load RNA Induced Silencing Complex (RISC) that represses mRNA translation. Bind to mRNA in sequence specific manner. Cause endonucleolytic cleavage of mRNA or block translation. Biomarkers of malignancies.
Silent Mutation	Synonymous mutation. Substation of single nucleotide by another. Protein product is same amino acid.
Promoter Mutation	Substitution of one nucleotide for another. Non-synonymous. Leads to altered gene expression.
Frameshift Mutation	Insertion or deletion of nucleotides that is not a multiple of 3. Likely to result in premature termination with loss of function or expression.
Dynamic mutation	Expansion of trinucleotide repeat. Leads to altered gene expression or altered protein stability or function.
Frequency of different mutation types	56% missense or nonsense 24% indels 10% splicing 7 % gross deletions or insertions 2% regulatory <1% other (complex rearrangement, repeat variation)
Synonymous Mutations	Mutation does not alter the polypeptide product of the gene.
Non-Synonymous Mutations	Leads to alteration in the encoded polypeptide.
Non-Conservative Substitution	Mutation codes for an amino acid that is chemically dissimilar, for example with a different charge. Can lead to reduction or loss of biological activity.
Conservative Substitutions	Single base-pair substitutions resulting in the replacement of a different amino acid that is chemically similar with no functional effect.
Nonsense-Mediated Decay	mRNA transcribe with premature termination codons are frequently degraded. RNA surveillance ?evolved to protect the body from the possible consequences of truncated proteins interfering with normal function.
Cryptic splice sites	Resemble the sequence of authentic splice site. May be activated when the conserved splice sites are mutated.
Exon Splicing Enhancer	Purine-rich sequences required for correct splicing of eons with weak splice-site consensus sequences.
Loss-of-Function	Reduced activity (hypomorph) or compete loss of the gene product (null allele, amorph).
Gain-of-Function Mutations	Increased level of gene expression or development of new functions of the gene product.
Dominant-Negative Mutations	Mutant gene in the heterozygous state results in the loss of protein activity, and consequently interfere with the function of the normal gene product of the corresponding allele.
Ionizing Radiation	Electromagnetic waves of very short wavelength and high-energy particles.
Dosimetry	Measurement of radiation. Dose expressed in relation to amount received by gonads. Often expressed as amount received in 30 years.
Translesion DNA synthesis	DNA replication machinery bypasses sites of DNA damage, allowing normal DNA replication and gene expression to proceed downstream.
Recombinant DNA	Union of two DNA fragments from different sources
Vector	Carrier DNA molecule used in the cloning process. Through independent replication within a host organism, allows production of multiple copies of itself. 5 types commonly used: plasmids, bacteriophages, cosmos, BAC, YAC
Restriction Enzymes	Enzymes that cleave double-stranded DNA in or near a particular sequence of nucleotides. Recognize palindromic nucleotide sequence of DNA between 4-8 nucleotides in length.
complementary DNA (cDNA)	DNA made by the action of the enzyme of reverse transcriptase on mRNA
Taq DNA polymerase	heat-stable DNA polymerase isolated from bacterium Thermophilus aquaticus. Used in PCR. Generates PCR products in matter of hours.
Southern Blotting	DNA digested by restriction enzyme. DNA fragments separated by size by electrophoresis on agarose gel. DNA fragments then denatured with alkali making them single stranded. Single-stranded fragments transferred onto nitocelulose filter (Southern Blot). DNA fragment of interest can be identified by adding radioactively labeled homologous DNA and then doing autoradiography.
Northern Blotting	mRNA is used as target nucleic acid. mRNA is unstable because of intrinsic cellular ribonuclease, so ribonuclease inhibitors can allow isolation of mRNA that can be run on a electrophoretic gel and transferred to a filter. Hybridizing the blot with a DNA probe allows determination of size and quantity of mRNA transcript.
Functional Cloning	Identification of a human disease gene via knowledge of protein product. The AA sequence of protein is used to make oligonucleotide probes to screen cDNA liberties.
Transcription Factors	Control RNA transcription from the DNA template by binding to specific regulatory DNA sequences to form complexes that initiate transcription by RNA polymerase
Zinc Finger	finger-like loop projection consisting of a series of 4 AA that form a complex with a zinc ion. Act as transcription factors through binding of zinc fingers to DNA.
CpG Islands	High concentration of dinucleotide CpG. Binding sites for transcription factors. Targets for DNA methylation.
CRISPR/Cas9	Clustered regularly interspaced short palindromic repeats. Widely used for genome editing. Engineered endonucleases with DNA-binding domain that will recognize a specific sequence in the genome (i.e. containing mutation), a nuclease creases double-stranded break, and homology-directed repair then repairs the break based on wild-type DNA template introduced into cells before editing. Concerns - off-target effects
Stop Codon	UGA, UAA, UAG

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