Genetics: Techniques in Molecular Genetics

Question 1

Gold standard test for looking at movement in genome (translocation)

Answer 1

Karyotype Analysis

Question 2

Karyotype analysis

Answer 2

Looks at entire genome, great for translocations (especially balanced), but limits of resolution (< Mb) is a problem. Also called chromosome analysis/cytogenetic testing.

Question 3

FISH

Answer 3

Allows you to look at one part of the genome in detail; you need to know which part. Fluorescently labelled probe hybridized overnight to target DNA sequence to identify sequences on metaphase chromosomes (lymphocytes, amniocytes, fibroblasts, chorionic villi, solid tumors, bone marrow aspirates), interphase cells (bone marrow, unstimulated peripheral blood, lymphocytes, buccal cells), tissue sections, direct preps (uncultured cells - amniotic fluid, smears - blood, bone marrow, urine, touch preps - tissue). Allows you to see: deletions, marker chromosomes, duplications, subtle translocations, analysis of non-random rearrangements in cancer, mosaicism quantification.

Question 4

Cytogenetic Testing

Answer 4

Karyotype analysis, ACDH for genomic syndromes. Indications include: prenatal diagnosis, preimplantation genetic diagnoses, infertility, recurrent miscarriages, stillbirths, confirmation/exclusion of chromosomal syndrome, abnormalities of sexual differentiation and development, developmental delay, mental retardation, dysmorphic features, neoplastic conditions (cancers)

Question 5

Arms of genetics

Answer 5

1) Congenital: inherited, 2) somatic alterations: genetics that occur through accumulation of mutations (ex. cancer)

Question 6

Advantages of FISH

Answer 6

Rapid technique (couple hrs to overnight), efficiency of hybridization and detection is high, large numbers of cells can be analyzed (40 - 100), study of interphase nuclei is possible, correlation of morphology and cytogenetics

Question 7

Disadvantages of FISH

Answer 7

Most FISH probes are not FDA approved (responsibility of each lab to validate each probe: sensitivity, specificity, normal ranges, expense), absence of detectable FISH deletion/duplication does not exclude a clinical diagnosis, need to know exactly what you want to look at

Question 8

Microarray technology/ array CGH

Answer 8

Compares genome of patient to a control/sample to evaluate copy number abnormalities in a single analysis. Targets specific regions of DNA to bind to with a labelled probe. Smallest probe is ~500 bp but it varies. Integrates molecular and traditional cytogenetics; not dependent on culturing cells for metaphase spreads.

Question 9

CNAs/CNVs

Answer 9

Look for regions of the genome where an individual has losses or gains of genetic material with no phenotypic impact at all (benign). Can have more than 2 copies of a chromosome. Creates challenges for microarray analysis.

Question 10

Types of microarrays

Answer 10

CNAs (look for losses/gains in genome, based on BACs/oligonucleotides as probes - ~60 bp), expression/cDNA arrays (look at differences in mRNA), SNP arrays (give copy # info and info about SNPs, parental origin), tumor microarrays

Question 11

For uniparental disomy should do…

Answer 11

SNP array

Question 12

Oligoarray analysis does not detect

Answer 12

Balanced rearrangements, uniparental disomy, imabalances in regions not included on the array platform, mutations within a gene, methylation changes. Sensitivity may be reduced by mosaicism (multiple cell lines in same individual); CNVs complicate interpretation. Limits of resolution: ~10,000-30,000 bps.

Question 13

PCR

Answer 13

Enzymatic method to copy a region of DNA

Question 14

Components of PCR

Answer 14

Enzyme, primers, nucleotides

Question 15

Enzyme used in PCR

Answer 15

DNA polymerase

Question 16

Primers

Answer 16

Small segments of DNA (~20 bp) used to target a region of interest. Hybridizes to template based on sequence.

Question 17

Purpose of nucleotides in PCR

Answer 17

To add to end of primer and grow DNA strand

Question 18

Microsatellite Analysis

Answer 18

Application of PCR. Used for human identity testing, paternity testing, bone marrow transplant monitoring. Looks for matches in microsatellites.

Question 19

Microsatellites

Answer 19

STRs. Will typically have ~13 in a genome.

Question 20

STRs

Answer 20

Specific sequence of DNA bases which contains repeats. # of repeats vary from person to person and are population specific.

Question 21

Types of STRs

Answer 21

Mono (AAAAA or CCCCC), Di (ACACAC or GTGTGT), Tri (ACTACTACT), larger (GATTACAGATTACA)

Question 22

PCR Limitations

Answer 22

Repetitive regions hard to amplify, SNPs can cause a problem (ex. differences in single bases near primer region), deletions can be a problem

Question 23

Sequencing DNA

Answer 23

Examining a region of DNA base by base. Ideal for detecting single base changes. Process: similar to PCR; Big Dye Sequencing using nucleotides with fluorescent labels, excite with laser and detect. Errors associated so may need to combine with other techniques.

Question 24

DNA sequence for heterozygote will display:

Answer 24

2 different peaks at that base

Question 25

MLPA process:

Answer 25

Uses probes throughout genome, ligates together, and amplifies.

Question 26

MLPA detects:

Answer 26

Small deletions/duplications of 1 exon, multiple exons, or the entire gene

Question 27

MLPA does not detect:

Answer 27

Single base changes, deletions/duplications larger than the gene or smaller than an exon, epigenetic changes

Question 28

De novo mutations

Answer 28

Occur in utero, may have ~60 in each person. Most benign, possible that some may cause disease (ex. neuro-psych conditions such as schizophrenia).

Question 29

Old method for detecting deletions/duplications:

Answer 29

Southern blot