1.  What do pharmacogenomic studies demonstrate?

2.  Define polymorphism

1.  That genotypic variation can effect phenotpyic response

2.  Genetic variation that exists in greater than 1% of the population (<1% is still a variant, but it is a non-polymorphic variant)

1.  SNPs

2.  Indels

3.  RFLPs

4.  STRs

5.  Randomly amplified polymorphic DNAs

SNPs

1.  Abbreviation for what?

2  Define

3.  How are they identified?

1. Single nucleotide polymorphism

2.  Single base pair substitutions, insertions, and deletions

3.  Sequence analysis 

Indels

1.  Define

RFLP

1.  What does abbreviation mean?

2.  Definition

1.  Restriction fragment length polymorphisms

2.  Nucleoside changes that can be analyzed using restriciton endonucleases as in RFLP analysis

STRs

1.  What does abbreviation mean?

2.  Definition

1.  Tandem repeat sequences (short tandem repeats)

2.  Small portions of DNA sequences repeated variable number of times (~1-20 bases)

Randomly amplified polymorphic DNAs

1.  Define

SSCP

1.  Abbreviation means?

2.  Define

3.  Used to screen what...compared to what?

4.  What type of test and what are you looking for?

1.  Single strand conformational polymorphism

2.  Method for identifying differences among known and variant DNA sequences WITHOUT SEQUENCING

3.  Used to screen PCR products from test samples for DNA variations compared with "normal" DNA

4.  PCR products migrate differently on a gel and the rate of their migration depends on conformation rather than size

RFLP

1.  Another method for identifying variant DNA without what?

2.  Reference is?

3.  How it works at a molecular level

4.  Migration depends on what and how does cost compare with SSCP?

1.  Sequencing

2.  "Normal" DNA

3.  PCR products with different sequences digested by restriction enzymes differently than PCR from "normal" samples

4.  Migration depends on fragment size

*Cost less than SSCP and can analyze longer fragments

Polymorphisms by Microsatellite

1.  Define microsatellite

2.  One of most abundant class of?

3.  Most important markers for (2)

4.  Used to generate?

1.  Repetitive sequences  (tandem repeats) of DNA composed of 1-5 bp units

2.  Intergenic repetitive sequences dispersed in human genome

3.  Gene mapping; Linkage analysis studies

4.  Comprehensive human genetic linkage maps

Detecting Polymorphisms by Microsatellite

1.  Most common repeats with denotation

2.  What length of repeats most commony used for polymorphism detection?

3.  What is critical one for Huntington's disease?

4.  Describe what size differences on gels mean

1.  Dinucleotide, esp CA...denote CAn where n = # of repeats

2.  Di, tri, and tetranucelotides

3.  CAG36-120

4.  Dinucleotide repeat...person with 14 repeates would produce fragment 2 nucleotides longer than someone with 13 repeats who would be 4 longer than someone with 11 repeats

Microsatellie Taq Polymerase

1.  Define TDT activity

2.  Define Slippage

3.  Way around these 2 things

1.  Terminal deoxynucleotidyl transferase activity = ability to add deoxynucleotides onto the end of an elongating DNA strand

2.  Polymerase slips during amplificaiton or replication resulting in insertions or deletions (esp common in sequences with repeats)

3.  Addition of T4 DNA polymerase which has 3' exonuclease activity

Microsatellites

1.  Stable or unstable within individuals?

2.  MSI means what and is a biomarker for what?

3.  What is the particular marker they are looking for and what does that marker do in a normal cell?

1.  Stable

2.  Microsatellite instability:  biomarker for cancer

3.  DNA mismatch repair (MMR) enzyme which produces a hypermutable state in the cell b/c MMRs do not correct replication mistakes leading to persistent mutations

MSIs as biomarkers

1.  What % of sporadic colorectal cancers (CRC) exhibit MSI

2.  What % of those are hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome

1.  15%

2.  ~5%...95% of all CRC tumors in HNPCC exhibit MSI

National Cancer Institute MSI as Biomarker

1.  How many are in the NCI panel?

2.  Whate are the 3 genotypic groups?

3.  Genotypes exhibit different

1.  5

2.  MSI-High:  2-5 panel loci unstable

MSI-Low:  1 panel loci unstable

MSS (MSI stable):  No MSI in any of 5 panel loci

3.  Phenotypes

1.  Legacy of scienticts of our time will be what?

2.  Improvement in time to analyze genes

1.  Development of field of molecular medicine that emerged fro the Human Genome Project

2.  DAys to sequence a single gene to hours to examine entire genome

PCR

1.  Uses what to make what?

2.  Denaturation temp; primer annealing temp; extension temp

3.  2 Ss and 1 R

1.  Polymerase, primers, and NTPs to make copies of DNA EXPONENTIALLY

2.  94C, 50-70C, 72C

3.  Simple, sensitive, reliable

PCR

3 uses for research and disgnostics with descriptions

1.  Virus detection:  tests for viral sequence rather than Abs, especially important for newborns where Abs may be maternal

2.  Forensics:  when only a small, non-reproducible sample of DNA evidence is available

3.  Deceased study subjects:  allows continued inclusion in studies with limited inital DNA samples

PCR Primers

1.  Length

2.  % of G+C content

3.  C&G balance

4.  Do you want repeats?

5.  What should binding sequence be unique for?

6.  Annealing temp

1.  21-24 bp

2.  40-60%

3.  Relatively equal

4.  No single base repeats

5.  Target gene of interest

6.  50-70C

PCR Polymerase

1.  Must be what in order to work in thermocycler

2.  Original one came from

3.  What was isolated from the same organism and is still widely used

4.  Sources of modern polymerases (2)

5.  Proofreading ability (_' -> _')

6.  Rate of error Taq and Pfu

1.  Thermostable

2.  Thermus aquaticus (hot springs)

3.  Taq

4.  Isolates or genetic engineering 

5.  3'-->5' exonuclease

6.  Taq:  1/100,000

Pfu:  1/1,000,000 

Direct DNA Sequencing

1.  How were PCR-amplified sequences separated?

Sanger Method for Direct DNA Sequencing

1.  AKA

2.  Uses what in addition to dNTPs

3.  How are #2 different from dNTPs

4.  Why include dNTPs?

5.  How dou tell which fragment terminates?

1.  Chain termination method

2.  2',3'-dideoxynucleotide triphosphate (ddNTPs)

3.  Have hydrogen at 3' carbon so cannot undergo elogation

4.  About 1% of reaction mixture is dNTPs to ensure growth of chains which then terminate at various lengths for sequencing

5.  Label primer or one of dNTPs with radiolabel or fluorescent label

Sanger Sequencing

1.  Why is flourescent labeling increasingly used (2)

ESTs

1.  Define

2.  What paradigm

3.  Processes developed to identify what?

1.  Expressed Sequence Tags 

2.  Gene-to-function-to-potential drug paradigm

3.  New molecular targets for drug discovery

Expressed Sequence Tags (ESTs):  Old way of drug discovery

List the 4 steps

1.  Disease cause identified:  mutation, toxin, infection

2.  Biological pathways examined for "broken cog" (enzyme/receptor malfunctioning)

3.  Enzyme or receptor identified and characterized through cloning/expression vectors

4.  Drugs designed to target enzyme/receptor to restore or replace function

Expressed Sequence Tags:  New approach 

1.  High throughput screening lead to what paradigm b/c you can rapidly identify thousands of genes with unknown function

2.  What are drug discovery teams using ESTs to do?

1.  Gene-to-screen paradigm

2.  Identifying genes that encode proteins that are attractive therapeutic targets

3.  ~100,000 genes in human genome expressed, but that represents only 5% of the genome and we curently lack technology to distinguish coding from non-coding nucleotide sequences

Expressed Sequence Tags (ESTs)

Method

1.  mRNA converted to cDNA w/ reverse transcriptase

2.  cDNA cloned into plasmids and cDNA library constructed

3.  Automated sequencing produce short DNA sequences from individual clones (These are the EST!!!) which are entered into a database and matched with known protein sequences

4.  Some ESTs long enough to suggest protein function alone

5.  Others you need to combine overlapping segments to piece together whole gene:  combined sequences called clone contig (contig short for contiguous sequence)

TIGR

1.  Stands for

Blots and what they analyze

1.  Southern

2.  Northern

3.  Western

4.  Eastern

1.  DNA

2.  RNA

3.  Protein

4.  Does not exist

Southern Blot

1.  Examines what?

2.  What enzymes are used?

1.  DNA

1.  Restriction endonucleases which regcognize specific dsDNA portions (restriction sites) and cut each strand at that site

Southern Blot

Method

1.  Extract DNA from cells and incubate/cut with restriction enzymes and denatured (some do not denature)

2.  Electrophoresis and transfer of DNA to membrane

3.  DNA detected using complementary DNA probe (Southern hybridization)

4.  Hybridization followed by prove detection procedure

Southern Blot DNA Preparation

1.  Method

1.  DNA extracted from cells

2.  DNA chopped up by restriction enzymes

**Note:  restriction enzymes will only recognize "normal" DNA so if there are mismatches or mutations, it will not work

3.  dsDNA denatured to ssDNA and stained with ethidium bromide for visualization under UV light

1.  Does DNA migrate toward anode or cathode

2.  What determines rate of migration?

1.  DNA migrates toward positive cathode since it is negative

2.  Size of DNA fragments

Southern Blotting

1.  What is the membrane usually made of?

2.  When do you stop calling it a gel and start calling it a blot?

3.  What was the most commonly used radioisotpes in the past?

4.  What is used today?

5.  2 modes of developing

1.  Nitrocellulose or nylon

2.  When the DNA is on the membrane

3.  32P

4.  Non-radioactive probes (dyes or chemiluminescent)

5.  Film or digital (more expensive so fiml still commonly used)

Southern Blot Molecular Wt Standards

1.  What 2 are common and for what size DNA

1.  Lambda/HindIII:  Large DNA fragments

PhiX174/HaeIII:  Small DNA fragments

Identify the following blots

1.  Southwestern

2.  Far Western

1.  Protein-DNA

2.  Protein-protein

RFLP: 

How is it like and dislike from Southern

1.  Alike:  Uses restriction enzymes

2.  Unalike:  No blotting b/c no probe necessary

Single starnd conformation polymorphism analysis

Difference from Southern blot

No restriction enzymes and DNA separated based conformation in SSCP and not size

*Important b/c one nucleotide may cause different conformation and no concern for accuracy of restriction enzyme

Vectors

1.  Define

2.  What do they allow for?

3.  Define expression vector

1.  A structure with DNA that can replicate autonomously and from which specific DNA sequences can later be isolated in pure form

2.  Allows for rapid and large production of copies of DNA sequences and the gene products from that DNA

3.  A research tool in which a gene is inserted in bacteria, yeast, algae, or other DNA and replicated under various condition to altern mRNA or protein EXPRESSION

Genetic Library

1.  What was it formerly known as

2.  Define cDNA

3.  Define Genetic Library

1.  DNA libraries, but there are now RNA, and methylated DNA libraries

2.  DNA made from RNA without introns

3.  Refers to large, documented, stockpiles of specific cDNA and vector collections from which researchers may draw and use in different studies (allows for direct comparision of resutls with known characteristics of the DNA in the library

Reverse Transcriptase

1.  Use

PCR

1.  Define oligonucleotide primers

2.  How many primer required

1.  Short DNA sequences that are complementary to a short sequence upstream of the sequence of interest

2.  2, one for sense and one for antisense

Stem Cells

1.  Define

1.  Cells that are the "stem" of differentiation...they have the genetic capability to create any type of cell and/or tissue in the body from which it originates (totipotent)

*come from in vitro

Stem cells

1.  What is our best guess as to why they differentiate the way that they do?

2.  Current use

1.  Differences in cytoplasmic environments since their DNA is identical

2.  Bone marrow transplantation for leukemia or lymphoma

Gene Therapy

1.  Define

2.  Only type of cell that it can be ethically used on?

3.  Currently used thearpies (3)

1.  Delivery of functional copies of a relevant gene to targeted cells to correct or repair a DNA mutation

2.  Somatic cells

3.  B-Thalassemia where normal DNA inserted into bone marrow to cure; Tumor necrosis gene inserted into tumor to kill tumor cells w/o hurting surrounding tissue; Angiogenesis genes inserted into heart tissue after hearet attack to regrow blood vessel

Gene Therapy

1.  What prevents insurance companies from using genetic tests against you?

2.  Major ethical issues with DNA

1.  Genetic Information Nondiscrimination Act (GINA) or 2008

2.  Can drug companies patent a person's DNA if they are the first with that sequence and it can be developed into a test?

Genetic Counseling

1.  What does it combine?

1.  Provision of risk information to individuals and families with appropriate psychological and educational support

Want to prevent something like Huntingtons related suicide through proper psychological training and means of support 

1.  Simple, easily applied, adaptable

2.  Can identify people at high and moderate risk

3.  Can be used in combination with other risk factors

4.  Useful for targeting interventions

5.  Positively influences healthy behaviors

Using FH for disease prevention

What is the proper intervention for someone with average, moderate, and high risk classifications?

1.  Average:  Standard prevention recommendations

2.  Moderate:  Personalized prevention recommendations

3.  High:  Referral for genetic evaluation and personalized prevention recommendations 

Suggested Guidelines for Risk Stratification based on FH

7 scenarios with relatives that can put you in a high risk stratification

1.  Premature disease in 1st degree relative

2.  Premature disease in a 2nd degree relative (CAD only)

3.  2 affected 1st degree relatives

4.  1 1st degree relative with late or unknown disease onset and an affected 2nd degree relative with premature disease from same lineage

5.  Two 2nd degree maternal or paternal relatives with at least one having premature onset of disease

6.  3+ affected maternal or paternal relatives

7.  Presence of a "moderate risk" family history on both sides of the pedigree

Suggested Guidelines for Risk Stratification based on FH

2 scenarios resulting in moderate risk stratification

1.  1 1st degree relative with late or unknown onset of disease

2.  2 2nd degree relatives from same lineage with late or unkonwn disease onset

Suggested Guidelines for Risk Stratification based on FH

4 things that earn an average risk stratification

1.  No affected relatives

2.  Only 1 affected 2nd degree relative from one or both sides of the pedigree

3.  No known family history

3.  Adopted person with unknown family history

Pedigree Analysis

1.  Use

2.  What info do you need (8)

3.  Collect on who and for how many generations

1.  To determine the mode of inheritance for phenotypic traits or diseases

2.  Ages, sex, ethnicit, general health status, major illnesses (chronic), cause of death, age of death, age when first dx

3.  First degree relatives (parent, sibling, offspring) for 3 gens

Characteristics of autosomal dominant inheritance

1.  Verticle or horizontal

2.  Heterozygoes express what phenotype

3.  Male:Female ratio

4.  How many parents must be affected

5.  % change your offspring has the disorder

6.  Frequency of sporadic cases positively assocaited with?

7.  3 characteristics common in phenotypes

1.  Verticle

2.  Abnormal phenotype

3.  Male to female is equal

4.  Only one parent must have it

5.  50% regardless of parental sex

6.  Severity of phenotype

7.  Less severe than autosomal recessive; assocaited twith malformations or physical features; age dependent

Characteristics of Autosomal Recessive Inheritance

1.  Veritcle or horizontal

2.  Male:Female

3.  Inheritance from parents

4.  % of affected, carrier, normal

5.  What fraction of clnically unaffected offspring are carriers

6.  What if individuals with same recessive phenotype mate?

7.  What if affected mate with non-carriers

8.  As phenotype gets more rare, what is likely?

1.  Horzontal with a single generation being affected

2.  Male and femal equal in frequency and severity

3.  Both parents must be carriers who are typically not affected by disease

4.  25%, 50%, 25%

5.  2/3

6.  All their kids will be affected

7,.  All children will be carriers

8.  Parents are consanguineous (related)

Characteristics fo X-Linked Inheritance Patterns

1.  What phenotype is impossible

2.  What are daughters of affected males

3.  Male:Female

4.  Whether heterozygous feamle counted as affected, and whether phenotype is called recessive or dominant depends on

1.  Male-to-male transmission phenotype

2.  All daughts are heterozygous carriers

3.  More men than women

4.  Sensitivity of the assay or examination

Characteristics of X-linked inheritance patterns

1.  When can homozygous mothers have an affected male child

2.  Proportion of heterozygous (carrier) moms negatively associated with

3.  What % of sons affected if mom heterozygous

4.  What can give the false impression of male-to-male transmission

1.  Germinal mutation

2.  Severity of condition

3.  50% of sons, 50% of daughters are carriers

4.  Affected male mating with heterozygous female b/c 50% of male kids will be affected

*In this case, 50% of females also affected and this may simulate autosomal dominant inheritance

Characteristics of Mitochondrial Inheritance

1.  Passed from who, to who

2.  Can men pass it along

3.  What % of offspring get it?

4.  What affects severity?

1.  Female to all offspring (including males)

2.  No

3.  100% b/c all ovum have the mitochondrial defect

4.  # of mutant mitochondria which can lead to heteroplasmy and unaffected transmitting females

Mutation

1.  Definition

2.  3 Classifications

1.  Any change in nucleotide sequence or arrangment of DNA

2.  Altering individual genes

Affecting number of chromosomes in a cell

Altering the structure of chromosomes

An all-or-none term indicaing the frequency of expression of a genotype

*When a pt has the mutation, how often do they have the disease (ex.  MMR mutation carriers have 80% chance of developing a Lynch syndrome cancer in their lifetime)

The extent to which a trait is manifest.  That is, the trait can vary in degree of expression from mild to severe

*When a pt has the mutation and expresses the disease, to what degree does he/she have the disease...ie, how bad

(ex:  pt with myotonic dystrophy express myotonia (prolonged muscle spasms), muscle weakness, and cataracts, but vary in # and severity of complicaitons b/t pts, even within same family

1.  Define:  Gene Dosage

2.  How is this overcome?

1.  X has more genes than Y, meansing women have twice as many gene, thus the potential for 2X as many gene products

*Represents potential survival advantage for women

2.  X-inactivation:  one of the X chromosomes inactivated by chance (Proposed by Mary Lyon)

*Occurs early in embryogenesis b/c all progeny contain the same inactivated X

X-inactviation

1.  What would happen if cells divide prior to X inactivation (2)

2.  Explain skewed X-inactivation

1.  Either both X's active or the opposite X active from the majority

*Females can have mosaic maternal and paternal X-expression

**Males must express the one X they have and all diseases with it

2.  Inactivation not complete or random usually...can produce clinically relevant phenotypic variation

1.  Autosomal aneuploidy

2.  X-Chromosome Aneuploidy

1.  Extra or missing autosomal chromosome

2.  Extra or missing X chromosome

*Phenotype may be normal or only mild disease compared with autosomal

*Assumed to be d/t:  over-inactivation and/or variation in time to inactivation during embryogenesis

Define Karyotypic Notation

1.  Normal Male

2.  Normal Female

3.  Turners syndrome (monosomy)

4.  Trisomy:  Klinefelter, Triple X, Trisomy 21

1.  46 XY

2.  46 XX

3.  45 X

4.  Klinefelter:  47 XXY

Triple X:  47 XXX

Trisomy 21:  47 XY +21 or 47 XX +21

mtDNA

1.  Where are most mitochondrial genes found?

2.  Only inherited from?

3.  # of disease identified in humans

4.  mtDNA replication

1.  Nuclear genes

2.  Mother

3.  40

4.  Cell makes sevearl copies of mtDNA prior to division and chance determines # of copies (mutated or WT) that progeny recieve (HETEROPLASMY)...if all mutated or all normal (HOMOPLASMY)

*Odds of homoplasmy much lower than nuclear DNA homozygosity 

mtDNA

1.  Heterogeneity in a phenotype is due to?

2.  What can happen as you age?\

3.  Most likely tissue affected:

1.  Heterogeneity in a genotype

2.  Bad mitochondria start to accumulate (possible role Parkinson's and Alzheimer's)

3.  High energy tissue:  muscle, brain, heart...usually progressive invovling many organ systems including the pancreas

*Hannah Poling compensated through federal vaccine injury fund d/t autism-like Sx that may have been the result of an underlying mitochondrial disorder

Phenomenon observed in family studies in which phenotype appears progressively "worse" in subsequent generations

*At younger age

With increase severity

Rate of disease pgoression may increase

combination of effects may occur

Genetic Anticipation

1.  Mostly seen where?

2.  First disease noted in and waht causes it?

3.  Other diseases (3)

1.  Dominantly inherited neurological and neuromusclar diseases

2.  Myotonic dystrophy:  unstable DNA sequence (CCG11 vs CCG19)

3.  Huntington's, fragile X syndrome, Freidrich ataxia

Genetic Anticipation

1.  Which disease is maternal and which paternal inheritance

1.  Maternal:  myotonic dystrophy

Paternal:  Huntington

*Not same as X or Y linked b/c these are autosomal and can be passed from contributing parent to either male or female

Genetic Anticipation

1.  Repeat length

1.  Variation can interfere with normal meiosis by causing frameshifts and affecting neighboring genes (gene deletion, etc)

*Repeat lenght can vary from generation to generation producing variation in expressivity d/t misalignment and/or frameshift

Parent-of-origin diferentially effects phenotype of same genotype; genotype is the same, but phenotype is different when mutation is inherited fromt he mother than father

*Mutation on chromosome 15:  if from mom-->Angelman syndrome, if from dad-->Prader-Willi syndrome

Genetic Imprinting

1.  Why does it occur?

Both alleles come from 1 parent, non fromt he other, by inheriting both of a homologous pair or segments of a homologous pair

*Whole chromosomes may occur when 1 parent is trisomic and gamete gets 2 of 3 chromosomes during meiosis

Uniparental Disomy (UPD)

1.  What can lead to it occuring in segments?

1.  Chromosomal rearragmenets, translocations, or duplications

*CF occasionally occurs in a child with only 1 carrier heterozygous parent...when false paternity is ruled out, UPD can sometimes be the culprit