Lab And Diagnostics Ii

Front 1

Markers used in paternity testing

Back 1

usually VNTRs

Front 2

In testing for DMD or Becker, what is the first method used? What is the sensitivity?

Back 2

multiplex PCR--picks up 65% of DMD, 85% of Becker (deletions)

Front 3

Name some disadvantages of sequencing

Back 3

1. time consuming
2. expensive
3. IDs polymorphisms
4. some repeats make sequencing difficult
5. large deletions not found

Front 4

Name the cf polymorphism near deltaF508 taht has no phenotypic effect on CFTR

Back 4

F508C

Front 5

If a high-risk family tests negative by sequencing, what other method may be used and what will it detect?

Back 5

MLPA-detects deletions

Front 6

For conditions in which many genes need to be sequenced and a chip is not available, what methods may be used to prescreen for mutations?

Back 6

SSCP (single strand conformation polymorphosm), heteroduplex analysis

Front 7

Name 2 methods used for heteroduplex analysis

Back 7

DGGE (denaturing gradient gel electrophoresis)
DHPLC (denaturing high performance liquid chromatography)

Front 8

How many repeats in the Huntington gene is normal?

Back 8

26 or less

Front 9

How many repeats in the Huntington gene is intermediate?

Back 9

27-35

Front 10

How many repeats in the Huntington gene is considered 'mutant?'

Back 10

Over 35 repeats

Front 11

How many repeats are associate4d with juvenile-onset Huntington?

Back 11

More than 60 repeats.

Front 12

What formula is used to determine what percent of carrier couples will have n affected children?

Back 12

(p+q) to the n
p=3/4
q=1/4

Front 13

A couple, each carriers of Tay Sachs, has 6 children. What is the chance that 4 of the 6 are affected?

Back 13

see binomial expansion

Front 14

Problems with heteroduplex analysis

Back 14

1. Highly effective, but doesn't ID ALL mutations.
2. Time consuming b/c have to sequence what you find
3. expensive

Front 15

What is the coefficient of selection?

Back 15

s, loss of fitness.
s=1-f
For complete fitness (f=1), s=0, because selection isn't affecting it. For lethal condition, f=0, selection coefficient =1.

Front 16

Name 2 types of gene scanning. When would you use them?

Back 16

1. SSCP
2. heteroduplex analysis
When it's too hard or expensive to seq. all possible genes and there is no chip..can ID possible mutations and then go back and sequence

Front 17

Relationship between f and s.

Back 17

If fitness (f) is 0, selection is 1. If f is 1, selection is 0.
As fitness improves, s decreases.

Front 18

Name 3 techniques to assess mutations when you don't already know what they are

Back 18

Sequencing, SSCP, heteroduplex analysis

Front 19

Uses for DNA fingerprinting

Back 19

Identity, paternity, UPD, maternal contamination

Front 20

What does Haldane's rule say?

Back 20

For X-linked conditions, m=1/3s.
So for lethal conditions where s=1, 1/3 of all cases are due to new mutations.
So 1/3 of X-linked cases are new mutations.

Front 21

Difference between identical by state vs. identical by descent

Back 21

Identical by descent means the same mutation came from common ancestor. Can be detected if nearby markers are also the same.

Front 22

What is the coefficient of kinship (phi)?

Back 22

Probability that 2 individuals share a common mutation by descent.

Front 23

Describe MLPA

Back 23

Each exon has 2 probes that are designed to hybridize adjacently within exon. Also have tails that bind to pcr probes. Bind exon probes and ligate, then do PCR rxn and run gel. Shouls see 1 band per exon, darkness corresponds to copy number.

Front 24

What is fitness (F)?

Back 24

The probability of passing on a gene to the next generation

Front 25

Types of targeted microarray

Back 25

Single chromosome
Known microdeletions
Subtelomeres
Tumor supressor loci

Front 26

Advantages and disadvantages of whole genome microarray

Back 26

Advantages=most complete coverage
Research applications
Disadvantages=disregard of rellevan t loci
Info difficult to interpret

Front 27

Adva ntages and disadvantages of targeted microarray

Back 27

Adv=look at specific region, eliminate results of unk own significance, characterize/eliminate polymorphisms, generatge relevant results for diagnostics
Disadv=miss dosage alterations not included

Front 28

What is gene flow?

Back 28

When a population's alleles change due to migration, rather than in genetic drift, which is due to chance.

Front 29

Which of these cannot be picked up by targetged microarray?
Mosaic aneuploidy
Identification of marker chromosome
Unbalanced translocation

Back 29

Picks up all of these

Front 30

Name 4 methods used to ID deletions and duplications

Back 30

MLPA
Southern blot
RT-PCR
Microarray

Front 31

What is a LOD score?

Back 31

logarithm of the odds of linkage compared with no linkage

Front 32

Best method to detect trinucleotide repeat disorders

Back 32

Southern blot plus PCR

Front 33

What is meant by a LOD score of 1?

Back 33

evidence of linkage is 10X stronger than evidence of non-linkage

Front 34

Method used to diagnose DMD

Back 34

Multiplex PCR, possibly plus Southern blot

Front 35

Name 3 diagnostic techniques that you might use if you know exactly what mutation to look for

Back 35

ASO (allele specific oligomers)
OLA (oligonucleotide ligation assay)
MALDI-TOF

Front 36

If one family shows a LOD score of 3.5 for 2 loci, and another family has a LOD score of 4.2, what is the LOD score?

Back 36

Add them-3.5 + 4.2 =7.7

Front 37

What types of mutations can be tested with ASO?

Back 37

Small kown mutations, point mutations, small deletions or insertions

Front 38

In ASO, if testing a patient for a particular mutation on a dot blot, what controls are needed?

Back 38

Known affected, known carrier, known non-carrier, water blank if amplified by pcr

Front 39

How is heritability calculated using twin studies?

Back 39

concordance rate in MZ twins minus concordance rate in DZ twins X 2

Front 40

Incidence of cf in US

Back 40

1 in 3000

Front 41

If a group from a Pacific island establishes a new population in the US, will the 2 groups have the same or different heritability?

Back 41

Different, because the environments are different, and environment plays a role in heratibility

Front 42

What percent of those with cf have pancreatic insufficiency?

Back 42

85%

Front 43

Name some disorders that are still tested for with Southern blotting

Back 43

Fragile X, DMD

Front 44

In the delta F508 cf mutation, what happens to the sequence?

Back 44

3 nuicleotides deleted=in frame, just missing one AA (phe)

Front 45

Name some trinuceotide repeat disorders which are diagnosed by PCR

Back 45

Huntington, spinocerebellar ataxia, Freidrich ataxia, moytonic dystrophy, SMA (not fragile X, because may be too may repeats to amplify

Front 46

Name 2 types of markers that can be assessed by PCR/Southern for identity testing

Back 46

RFLPs, microsatellites

Front 47

What is the phenotype of the cf mutation F508C?

Back 47

Phenalyalanine changed to cysteine, which is much better than deleting it as in deltaF508. F508C is apparently benign.

Front 48

Microsatellites are often found where in the genome?

Back 48

introns

Front 49

OLA (oligonucleotide ligation assay) can be used only for identifying what types of mutations?

Back 49

Must be known, common mutations, and can only be single base substitutions or very small deletions

Front 50

Explain the principle behind OLA assays

Back 50

oligonucleotide ligation assay--uses 2 oligos per rxn. Template is either mutant or WT DNA, looking for a known point mutation or tiny deletion. One oligo is a probe to the mutant (or WT) area and has a tag, the other binds to the adjacent region and has a fluorescent label. Ligase is added, but ligation only occurs if both probes bound to the strand. Pull out tagged bits, wash, measure fluorescence. Will only have fluorescence attached to tag if both ligated.

Front 51

Name 3 types of microsatellites

Back 51

variable nucleotide tandem repeats (VNTR)
short tandem repeats (STR)
simple sequence repeat (SSR)

Front 52

What should you do if a mom is identified as a cf carrier, and dad cannot be tested (no insurance, unavailable, etc)?

Back 52

Offer amniocentesis. Risk to baby in this case is about 1%. Also look for echogenic bowel on ultrasound.

Front 53

Following a deletion found by microarray in a child, what 2 steps would you do next, and in what order?

Back 53

1. confirm with FISH
2. test the parents

Front 54

When a patient is confirmed to have a new deletion by microarray, what needs to be determined?

Back 54

Which genes are in the region, what their function is, and what it may mean for the patient and family

Front 55

When a child is found to have a deletion by microarray, how often is a parent found to have the same deletion?

Back 55

about 10% of the time

Front 56

In CGH, how often in a variant of unknown significance found?

Back 56

5-10% of the time

Front 57

Problems with analyzing fetal cells from maternal serum

Back 57

low yield, hard to isolate, poor FISH staining, no better detection rates than biochemical serum sreening
97.5% detection, 5% false positives)

Front 58

Up to how much of free-floating DNA in maternal blood is from the fetus?

Back 58

up to 11%

Front 59

When does an Rh- mom need Rhogam injections?

Back 59

after bleeding
after CVS/amnio
after delivery
at 28 weeks

Front 60

Treatment for an Rh- mom who is already sensitized

Back 60

1. Initial amnio to genotype fetus
2. Monitor with u/s or amnio every 2 weeks
3. If necessary, in utero transfusion or early delivery

Front 61

In screening for Rh D locus and SRY, what do the following results mean?
RhD+/SRY+
RhD-/SRY+
RhD+/SRY-
RhD-/SRY-

Back 61

RhD+/SRY+ =male at risk
RhD-/SRY+ =male not at risk
RhD+/SRY- =female at risk
RhD-/SRY- =female not at risk, OR error in test