Matise - Human Genome Karyotype

Question 1

ENCODE Project

Answer 1

Goal: discover “functional” DNA. Mapped human genome for transcript and coding exons, chromatin modification and DNA methylation, DNAase hypersensitivity, transcription factor binding….
Showed: Many promoters/enhancers in non-coded region, and much of it codes RNA for unknown function. But not fully accurate, some evolutionary baggage

Question 2

Repetitive Sequences (3)

Answer 2

1) Short Repeats: (micro)satellite sequences, variable repeats allows differentiation
2) Tandem Repeats: can be duplications of gene, increasing recombination opportunities and potential errors (red-green colorblindness), or deletions (diagnose with FISH probes)
3) Retrotransposons: >~25% genes, Alu sequences most abundant (contain Alu restriction site). RT insertion of mRNA into DNA can disrupt gene, causing disease

Question 3

Karyotyping Techniques (3)

Answer 3

1) G-Banding
2) FISH (Fluorescent in situ Hybridization)
3) CGH (Comparative Genome Hybridization)

Question 4

G Banding

Answer 4

Arrests cells in metaphase (colchicine), causing condensation, stain with Giemsa dye. Identify by size (1–>22 big–>small), centromere location, banding pattern.
Can only detect large structural changes (~45 genes). Need FISH/CGH for better resolution

Question 5

Chromosome centromere positioning

Answer 5

Metacentric - centromere in middle (p=q) ex: 3
submetacentric (ex 18)
acrocentric ex: 22
telocentric: centromere at end of chromosome (not found in humans)

Question 6

Reading Chromosome banding: 6q31.1

Answer 6

chromosome 6, q (long) arm, band 31.1

Question 7

FISH (fluorescent in situ hybridization

Answer 7

Fluorescent probe binds complementary DNA. Faster than G-banding, so used for neonate diagnosis (interphase faster>metaphase, but less resolution). Can easily see aneuploidies/monoploidies. CAN ONLY SHOW DELETIONS, NOT SNPs or rule out mutations in non-probe regions.

Question 8

CGH (Comparative Genome Hybridization)

Answer 8

Microarray of sequences complimentary to genome. PCR, compare patient DNA-control DNA binding for each oligonucleotide. Red-yellow-green, underproduction-normal-overproduction.
Advantages: non-specific, detects small changes
Limitations: Detects deletions/duplications but not inversions/translocations

Question 9

Aneuploidy

Question 10

Robertsonian Translocation

Answer 10

breakpoint = in centromere of D (13-15) and G (21-22) chromosomes. Often fuses q-arms, so lose p but not fatal. Designation: 45, XX, -14, -21, +rob(14q;21q). Only 2/6 chance viable kids.

Question 11

Pericentric Inversion

Answer 11

Includes centromere,

Designation: 46, XY, inv(6)(p23;q21)

Question 12

C-Value

Answer 12

Amount of DNA in one copy of genome. 3.2x10^9 in humans. Doesn’t correlate with organism complexity (nor does ploidy, # of chromosomes