CHAPTER 11: Analyzing Genomic Variation

difference in DNA sequences among individuals are called

DNA polymorphisms

because siblings, and also parents and children, share 50% of all _______ alleles, it is possible to identify the prepetrator of a crime not only by a match to his or her own DNA, but also by a ________ match to the DNA of a close relative

SSR; partial

Huntington's disease is caused by what type of genetic variant?

SSRs

what types of polymorphisms can always be genotyped by performing PCR and then separating the PCR products by gel electrophoresis (w/o sequencing)? SNPs SSRs CNVs DIPs

SSRs & DIPs

copy number variant (CNV)

a category of genetic variation arising from large regions (from 10 bp up to 1 Mb) of duplication or deletion.

polymerase chain reaction (PCR)

a fast and inexpensive method of replicating a DNA sequence when short sequences at each end are known; based on a reiterative loop that amplifies the products of each previous round of replication.

deletion-insertion polymorphisms (DIPs or InDels)

a genomic DNA polymorphism caused by insertion or deletion; in humans, they occur once in every 10 kb of DNA.

when knowing the mutation that causes a disease does not help in the treatment of the disease, the outcome is not ______

actionable

exome

all exons in a genome

lumacaftor

ameliorates the folding problem resulting from the ΔF508 mutation of the CFTR gene

if a genetic condition is recessive and the parents are not even distantly related, the patient might be a ___________ heterozygote

compound

patients with what disease are often compound heterozygotes for the disease allele?

cystic fibrosis

in PCR, primers bind to...

denatured (single-stranded) template DNA

nonanonymous DNA polymorphisms

differences in genomic DNA sequence that have an effect on gene function. (affect gene expression, and this affects phenotype)

T/F: a single fluorescently labeled DNA molecule generates enough light to allow its detection on a microarray

false

DNA _______ is performed by PCR amplifying 13 different SSR loci using 13 pairs of PCR primers with dyes that fluoresce in different colors

fingerprinting

disease genes

genes whose mutant alleles cause human genetic diseases

some uses for DNA fingerprinting

identification of human remains, forensics, and paternity suits

the association of a molecule marker that is transmitted along with a genetic disease in a family pedigree indicates that the molecular marker...

is likely to be close to the gene that causes the disease

identification of many disease-causing mutations depends on...

knowledge of variants from many other people's genomes

genetic variants that affect phenotypes through changes in protein function are called

nonanonymous DNA polymorphisms

in PCR there must be sufficient _______ triphosphates and oligonucleotide _______ in the reaction tube to support multiple rounds of DNA replication

nucleotide; primers

another name for the configuation of alleles is their

phase

a genetic variant involving the change of a single base is called a(n)...

single nucleotide polymorphisms (SNP)

primers are chosen for PCR based on...

the DNA sequence on both sides of the target region

DNA polymorphisms

two or more alleles at a locus. The sequence variations of a DNA polymorphism can occur at any position on a chromosome and may (nonanonymous polymorphism), or may not (anonymous polymorphism), have an effect on phenotype.

steps in one cycle of a PCR reaction

1. denaturation of target DNA into single strands 2. hybridization of primers 3. polymerization of DNA polymerase

an actionable outcome came from the bioinformatics analysis behind variations in what genes? CFTR DHOD XIAP

CFTR & XIAP

the defective _____ protein expressed in patients with the prevalent ΔF508 mutation cannot _______ properly

CFTR; fold

what new innovations are used in high-throughput sequencing? based addition is controlled temporally so that each base can be identified before the next one is added individual DNA molecules are synthesized by DNA polymerase anchored in one piece the ability to add complementary nucleotides to a template DNA strand without the use of DNA polymerase the sensitivity of detection is so high that a single molecule of DNA can be monitored without the need for cloning or PCR amplification the development of in vivo sequencing

based addition is controlled temporally so that each base can be identified before the next one is added individual DNA molecules are synthesized by DNA polymerase anchored in one piece the sensitivity of detection is so high that a single molecule of DNA can be monitored without the need for cloning or PCR amplification

after 32 cycles of PCR, a DNA sequence may be amplified a ____-fold

billion

one limitation to positional cloning is that you must be able to determine the _______ of the SNP and disease alleles for the people in the pedigree

phase

positional cloning tracks one locus by _______ and the second locus by direct _______ of each person

phenotype; genotyping

in 1985, Kary Mullis developed a way to copy DNA without vectors or host cell. this technique is called...

polymerase chain reaction (PCR)

short olgionucleotides that flank the region of DNA to be amplified by PCR are called

primers

simple sequence repears (SSRs) arise from

rare, random events

the mutation that causes sickle-cell disease is a ______ ________ polymorphism

single nucleotide

the technique of sequencing PCR products amplifies from genomic DNA can be used to genotype what type of variants: - small DIPs - aneuploidy - CNVs - SNPs - expansions/contractions of the number of repeats in SSRs

small DIPs, SNPs and expansions/contractions of the number of repeats in SSRs

nucleic acid hybridization

the ability of complementary single strands of DNA or RNA to come together to form double-stranded molecules, is the basis for many techniques in molecular biology

if we assume that the likelihood that two random people share exactly the same two alleles at a particular SSR locus is 10%, then the chance that two random people will have the same combinations of SSR alleles at 13 unlinked position is roughly one in....

( 0.1 * 0.1 = 0.01; (0.1^13)=) 10 trillion

place these steps in determining a Lod score in order from first to last: - calculate the likelihood ratio - interpret the Lod score - calculate the Lod score - tabulate which progeny are parental and which are recombinant - calculate the Lod score numerator and denominator

1. tabulate which progeny are parental and which are recombinant 2. calculate the Lod score numerator and denominator 3. calculate the likelihood ratio 4. calculate the Lod score 5. interpret the Lod score

in PCR, the template DNA is the....

DNA to be amplified

the sequencing of the entire genomes of all members of a nuclear family was first done in looking for the mutation that causes

Miller syndrome

order these categories of genetic variants in order from most to least frequent in the human genome: - CNV (copy number variant) - SSR (simple sequence repeat) - SNP (single nucleotide polymorphism) - DIP (insertion/deletion)

SNP (single nucleotide polymorphism) DIP (insertion/deletion) SSR (simple sequence repeat) CNV (copy number variant)

amniocentesis

a medical procedure in which a sample of amniotic fluid is taken from a pregnant woman to determine the karyotype of an unborn baby.

DNA fingerprint

a representation of parts of an individual's DNA that can be used to identify a person at the molecular level

microsatellites (short tandem repeats)

an SSR locus. The term derives from the fact that genomic DNA fragments containing SSR loci separate from fragments without repeat sequences in density gradient centrifugation experiments.

derived allele

an allele that arises through mutation.

DNA marker

an identifiable physical location on a chromosome with DNA sequence variants whose inheritance can be monitored.

compound heterozygote (or trans-heterozygote)

an individual who carries two different alleles at a locus resulting in a recessive phenotype

the National Center for __________ Information has a catalog of more than 50 million single nucleotide _________ found in the human genome

biotechnology; polymorphisms

in whole-exome sequencing, scientists first enrich for genomic DNA fragments that correspond to the exons of all genes by hybridizing to...

cDNA sequences

anonymous DNA polymorphisms

differences in genomic DNA sequence with no effect on gene function. ( affects neither the nature nor the amounts of any protein in the body)

what characteristics might a genetic variant responsible for a rare disease have?

different patients suffering from the same symptoms may share the same mutation

characteristics of the final PCR product

double-stranded and extends from one primer's 5' end to the position of the other primer's 5' end.

because whole-genome sequencing is still expensive, some researchers instead perform whole- ________ sequencing which focuses on the portion of the genomes corresponding to protein-coding exons.

exome

scientists searching for mutations that cause disease first look in the ______, because the DNA sequences that regulate transcription and splicing can be more difficult to identify

exome

what types of variants are likely to be nonanonoymous polymorphisms

frameshift mutations nonsense mutations missense mutations

simple sequence repeats (SSRs)

genomic locus that consists of one or a few bases repeated in tandem up to 100 times. Different alleles have different repeat numbers. The human genome contains ~100,000 SSR loci. Also called a microsatellite locus.

deafness and high blood pressure are conditions that display

locus heterogeneity

Lod score (log of odds ratio)

log of odds ratio; statistic used to analyze linkage data in humans. The statistic determines, for a particular data set, how much more likely it is that two loci are linked with a given RF value than that they are unlinked.

molecular markers can be used to identify the location of a disease-causing gene because they...

may be located close to a disease-causing gene

a Lod score is calculated from the _____ of the probability of obtaining a particular set of results in a ______ of two loci are linked to the probability of observing the same results if the loci are unlinked

ratio; pedigree

allele-specific oligonucleotides (ASOs)

short oligonucleotides that hybridize with only one of a pair of alleles distinguished by a single base difference

DNA microarray

silicon chips or microscope slides with DNA fragments that can allow many genes in a genome to be studied simultaneously

in PCR, DNA polymerase adds nucleotides to...

the 3' ends of primers

a Lod score is used in human genetics rather than a X^2 statistic because

the Lod score better handles a small number of data points while allowing the data obtained from many different pedigrees to be combined.

ancestral allele

the allele carried by the last common ancestor of two species.

problems that may impede a geneticist's ability to identify the mutation responsible for a disease

the mount of variation among human genomes is huge and no genome sequence is 100% accurate or 100% complete

why might a mutation remain unidentified even when a whole-genome sequence is available

the mutation may affect transcription of the disease gene and splicing of the disease gene's transcription without altering splicing consensus sequences

Combined DNA Index System (CODIS)

the national database containing the DNA profiles of individuals who are convicted of sexual and violent crimes

in a rare dominant condition it is likely that...

the patient will be heterozygous for the causative allele related patients will have the same rare mutant allele unrelated patients might have a different mutation in the same gene

steps of high-throughput, single molecule DNA sequencing - DNA polymerase synthesizes new DNA containing m=nucleotides with colored, base-specific fluorescent tags, and with blocks on their 3` end - single-stranded genomic DNA is attached to a flow cell - new DNA polymerase and labeled, blocked nucleotides are added to the flow cell, and the reaction is repeated about 100 times - a high-resolution camera photographs the flurosecence, and the tag and blocking groups are removed from the most recently added nucleotide

1. single-stranded genomic DNA is attached to a flow cell 2. DNA polymerase synthesizes new DNA containing m=nucleotides with colored, base-specific fluorescent tags, and with blocks on their 3` end 3. a high-resolution camera photographs the flurosecence, and the tag and blocking groups are removed from the most recently added nucleotide 4. New DNA polymerase and labeled, blocked nucleotides are added to the flow cell, and the reaction is repeated about 100 times

preimplantation embryo diagnosis

a process by which human eggs are fertilized in vitro and genotyped for particular disease alleles prior to implantation in a uterus.

single nucleotide polymorphisms (SNP)

a single nucleotide locus with two naturally existing alleles defined by a single base pair substitution. SNP loci are useful as DNA-based markers for genetic linkage analysis.

choose molecules that can undergo nucleic acid hybridization: - a double stranded DNA and a complementary single-stranded RNA - a single-stranded RNA and the protein it encodes - a single-stranded DNA and a complementary single-stranded RNA - two complementary single-stranded RNAs - two identical double-stranded RNAs - two complementary single-stranded DNAs

a single-stranded DNA and a complementary single-stranded RNA -two complementary single-stranded RNAs two complementary single-stranded DNAs

allelic heterogeneity

a situation where a genetic disease is caused by a variety of different mutations in the same gene.

locus heterogeneity

a trait where mutations in any one of two or more genes results in the same mutant phenotype.

reasons why very few difference in DNA sequences between two people alter the amino acid sequences of proteins... - all mutations within codons are repaired by DNA repair machinery - a very small percentage of the human genome consists of codons within genes - many mutations within codons are silent - mutations do not occur within coding sequences - natural selection might lead to the disappearance of deleterious mutations

a very small percentage of the human genome consists of codons within genes many mutations within codons are silent natural selection might lead to the disappearance of deleterious mutations

scientists utilize the close relationship between the human and _________ genomes to determine if an SNP is ancient, or of it is a derived allele resulting from a relatively recent mutational event

chimpanzee

the strategy in which the location of a gene associated with a hereditary disease is identified by finding polymorphic loci to which the mutation is genetically linked is called

positional cloning

positional cloning

the process by which researchers use linkage analysis to obtain the clone of a gene.

in 2003 the Human Genome Project sequenced the complete human genome at a cost of ______ dollars. In 2006, the cost was around _______ dollars.

three billion; two thousand

the first goal of positional cloning is...

to discover a DNA marker that shows linkage to the disease locus

positional cloning takes advantage of the fact that two loci that are genetically linked are..

found close together on the same chromosome

Prenatal genetic diagnosis

genotyping fetal cells to determine alleles of disease loci via methods like PCR

in positional cloning, at least one parent must be a double __________ for the cross to be informative

heterozygote

the ability to rapidly sequence large amounts of DNA is called

high-throughput sequencing

how might a non-actionable outcome of whole genome sequencing to identify a disease gene still provide useful information?

it may help patients to guide their reproductive decision and provide information that helps identify a future treatment for the disease

to conduct _________ _________ sufficient pedigree data must be available for the disease of interest

positional cloning

how can embryos produced by in vitro fertilization canmundergo genetic testing prior to being placed in the mother's womb

preimplantation embryo diagnosis

the CODIS database allow forensic laboratories throughout the U.S. to compare DNA profile. these laboratories use the same 13 _______ pairs to amplify 13 ______ sequence repeats

primer; loci