Genomics & Genetic Sequencing

Question 1

How frequent are SNPs?

Answer 1

~1 SNP per 1,000 nucleotides, between any two individuals

Any two genomes are separated by ~ 3 million SNPs - still 99.9% identical

Question 2

Genome composition

Answer 2

1.5% protein coding
5% regulatory (gene expression, development)
20-25% genes (including regulatory sequences)
40-50% repetitive DNA (total)
50% unique (single copy) DNA (total)

*does not add up to 100% because of reasons

Question 3

Short Interspersed Repetitive Elements (SINEs)

Answer 3

Ex: Alu family - ~300bp related members, 500,000 copies in genome

Question 4

Long Interspersed Repetitive Elements

Answer 4

Ex: L1 family - ~6kb related members, 100,000 copies in genome

Question 5

Satellite DNAs

Answer 5

Short repeat sequences organized tandemly (head-to-tail) and in clusters; comprise 10-15% of genome

Families vary with regard to location in genome, total length of tandem array, and length of constituent repeat units that make up the array

Question 6

Hot spots for human-specific evolutionary changes

Answer 6

Satellite DNAs, including a specific pentanucleotide sequence, found as part of human-specific heterochromatic regions on the long arms of Chr 1, 9, 16, and Y

Question 7

Alpha Satellite Repeats

Answer 7

171 bp repeat unit found near centromeric region of all human chromosomes; likely important for chromosome segregation in mitosis and meiosis

Question 8

Alu family

Answer 8

Short interspersed nuclear element (SINE); related repeats, ~300 bp long, dispersed throughout the genome; ~10% of genome

Question 9

L1 Family

Answer 9

Long Interspersed Nuclear Element (LINE); related repeats, ~ 6kb long, dispersed throughout the genome; ~20% of genome

Question 10

Pseudogenes

Answer 10

DNA sequences that closely resemble known genes/gene families but are nonfunctional, either as a result of inactivation mutations (nonprocessed) or by retrotransposition (processed)

Question 11

Retrotransposition

Answer 11

Process involving transcription, followed by reverse transcription and integration of that cDNA back into the genome; formation of non-functional, processed pseudogenes

Question 12

Single Nucleotide Polymorphisms (SNPs)

Answer 12

A difference in a single DNA nucleotide base, within a particular gene, that gives rise to 2 discreet alleles; allele frequencies differ in different ethnic groups/populations

Question 13

Simple insertion-deletion polymorphisms (indels)

Answer 13

Variation caused by insertion or deletion of segments between 2 and 100 nucleotides; gives rise to 2 discreet alleles - presence or absence of the inserted or deleted segment

Question 14

Short Tandem Repeat Polymorphisms (Microsatellites)

Answer 14

Stretches of repetitive DNA consisting of units of 2-4 nucleotides, repeated between 1 and 30 times; different alleles result from differing numbers of repeated nucleotide units; often used as a marker in forensics

Question 15

Minisatellites

Answer 15

A class of variable number tandem repeats (VNTR); results from insertion of varying numbers of copies of a DNA sequence 10-100bp in length; many alleles, due to variation in the number of copies of the tandem repeat

Question 16

Variable Number Tandem Repeats (VNTR)

Answer 16

Variation in the number of minisatellites that are repeated in tandem

Question 17

Copy Number Polymorphisms (CNPs)

Answer 17

Recurring deletions or insertions of larger sections of a chromosome, leading to gaps or duplications; may have two alleles (related to presence or absence of the copy) or multiple alleles (due to number of copies of the segment present)

Question 18

Short Tandem Repeat Polymorphisms (STRPs)

Answer 18

Different alleles resulting from variation in the number of nucleotide repeat units within a microsatellite region

Question 19

Polymorphic DNA markers

Answer 19

Scoreable differences at known genomic positions; most often microsatellites, SNPs, and CNVs

Question 20

Haplotype Blocks

Answer 20

10-50 kb chromosomal regions located in-between rearrangement hot-spots, where recombination is rare; SNPs and marker alleles within blocks tend to be co-inherited and are said to be in linkage disequilibrium (LD)

Question 21

Candidate gene association study

Answer 21

Hypothesis-driven approach relying on a priori biological or positional hypothesis; done by genotyping markers within a candidate gene and then comparing allele frequencies in cases versus controls; association does not prove causation by the associated variant but implies at least LD with casual mutation

Question 22

Population stratification

Answer 22

Genetic variation within grossly homogenous populations; variation may be linked to disease and therefore cause false positive results in case-control candidate gene studies

Question 23

Genetic-linkage studies

Answer 23

Used to study genome segments that are disproportionately co-inherited along with disease to determine if the loci are linked; “multiplex” families with multiple cases of a disease are studied to determine the frequency of recombination events between two loci of interest; a LOD score (logarithm of the odds) of +3 or greater is evidence that two loci are linked

Question 24

Genome-wide association studies (GWAS)

Answer 24

Tests many markers (SNPs) across entire genome searching for significantly different allele frequences in cases vs. controls; can identify many genes that contribute to a certain disease

Same as candidate gene case-control association study but tests MANY markers across entire genome; requires larger sample size (>1,000 cases/controls) to correct for multiple testing problem

Can discover new mutations that contribute to disease

Question 25

Chromosomal Analysis

Answer 25

Detects anomalies in chromosome number as well as large structural rearrangements (deletions, insertions, etc.) ex: Trisomy 21

Question 26

FISH

Answer 26

Flourescence In-Situ Hybridization; flourescently labeled, locus-specific probe (several Kb) can identify micro-deletions or multiple copies of loci of interest in patient DNA (isolated in metaphase or interphase) Detects chromosomal microdeletion/duplication, chromosomal rearrangements (in cancers), and gene copy numbers

Question 27

Chromosomal microarray analysis (CMA)

Answer 27

Test and reference DNA samples are labeled with different colors, mixed, and affixed to an array; oligonucleotide probes containing DNA fragments can examine many different loci simultaneously; abnormal ratios of the colors are indicative of deletions or duplications Cannot detect balanced translocations, inversions, or point mutations May also detect benign CNVs (vs. test DNA) creating "noise" on the array

Question 28

Diagnostic Testing

Answer 28

A positive genetic test result in a patient with signs or symptoms of a genetic disease can confirm an already suspected disease or diagnose the underlying and current disease

Question 29

Predictive Testing

Answer 29

A positive genetic test result in a patient with NO signs or symptoms of genetic disease provides estimate of future disease risk

Question 30

Chaperone-Based Therapy

Answer 30

Useful in genetic disorders characterized by a misfolded mutant protein; chaperone can help the mutant protein fold appropriately, restoring protein function Ex: Small percentage of Fabry disease patients benefit from administration of galactose, a chemical chaperone

Question 31

Retroviral Approach to Gene Therapy

Answer 31

Advantages: Vector can accomodate up to 8 kb of added DNA and the integrated DNA is stable Limitations: Target cell must undergo division for integration of recombinant DNA into host genome; limited use in nondividing cells (i.e. neurons)

Question 32

Adenoviral Approach to Gene Therapy

Answer 32

Advantages: Can accommodate inserts of 30 to 35 kb and infect dividing or nondividing cells Limitations: One case of strong immune response resulting in death

Question 33

Non-Viral approach to gene therapy

Answer 33

1. Naked DN?a (ex: cDNA with regulatory elements in a plasmid) 2. DNA packaged in liposomes 3. protein-DNA conjugates 4. Artificial chromosomes

Question 34

Positional cloning

Answer 34

Identifies disease genes by first determining the gene's location through linkage analysis, followed by attempts to identify the gene on the basis of it's map position

Question 35

Candidate gene sequencing

Answer 35

Hypothesis-driven approach depending on an a priori biological or positional hypothesis; gene is identified and sequenced directly

Question 36

Case-control genetic association test

Answer 36

Compares allele frequencies between cases and controls in an ethnically-matched sample; association does not imply causation by the associated variant but does suggest LD with a causal mutation Population stratification may lead to false positives