human genetics unit 1 - final

how many pairs of autosomes?

22

how many sex chromosomes?

2 (1 pair)

what are autosomes numbered in relation to?

size

chromosomes are composed of mixture of ____ and _____

DNA and proteins

what helps with DNA packaging

histones

each cell contains ___ (length) of DNA if stretched end to end

2 meters or 6 feet

the nucleus of a human cell is ____ in diameter

6 um

when is euchromatin loosened

during interphase

what percentage of the human genome exists as euchromatin?

92%, high gene density

what does heterchromatin contain

a lot of highly repetive sequences

what is function of heterochromatin? and how does it do it?

maintains stability by silencing repetitive DNA, repressing certain genes to retain cell identity and ensure proper organization of chromosomes during cell division

why was euchromatin the main focus when dna sequencing was first coming out?

it’s more useful, it has high gene density, it’s hard to accurately sequence the repetitive sequences in hetchromatin

what time period did HGP take place

1990-2003

what method was used in HGP

sanger sequencing

explain sanger sequencing

1. pcr reaction conducted with a lot of copies of DNA

2. dNTP’s incorporated to tag the nucleotides with their identity

3. ddNTP’s stop the reaction when they are used at random because it lost 3’-OH group so the base physically cannot be tagged

4. if have enough copies, eventually all spots will be tagged

5. conduct gel electrophoresis to analyze, read in bottom to top order and use fluorescently labeled tags to identify base

who and when invented sanger sequencing

frederick sanger, 1977

what two things did frederick sanger will nobel prize for

determining insulin sequence, sanger sequencing

what protein is responsible for DNA synthesis

dna polymerase

direction of dna synthesis

5’ —> 3’

what is happening in dna synthesis?

Each new nucleotide is added to the growing chain by linking the 5’ alpha phosphate of the incoming deoxyribonucleoside triphosphate to the 3’ hydroxyl group at the 3’ end of the growing chain

how do fluroescent dyes label nucleotides?

fragments share a common 5′ end sequence (defined by the primer sequence) but have variable 3′ ends due to ddNTP termination so the ddntp does it basically

capillary electrophoresis

long, thin, acrylic-fiber capillary; An electric current pushes the DNA fragments through the capillary; since the capillary is extremely small in diameter, the separation of bands is a lot faster

pitfalls of sanger sequencing

Limited throughput; sequencing one DNA fragment at a time

Max sequence fragment length is ~850 bp

Not cost effective

If sequencing lots of DNA fragments, DNA input is not a trivial amount

what were the two competing approaches when HGP

Human Genome Project (publicly funded), cloning DNA fragments and then mapping to determine sequence order vs. Private company (Celera), shotgun sequencing approach

HELP explain the HGP approach to sequencing in more detail

Bacterial and yeast artificial chromosomes

Cloning employed to generate ample DNA template for sequencing and aid with assembly

Sequenced clones were assembled based on overlapping base content

what is clone contig

set of overlapping cloned DNA segments that together provide a continuous physical map of a specific genomic region

explain the private company shotgun sequencing in detail

Large amounts of genomic DNA were fragmented

DNA fragments were sequenced and algorithmically assembled into long stretches of DNA sequence

Computation was a major issue for Celera; assembling the vast number of short DNA fragments proved to be a difficult problem, requiring Celera to build the most powerful supercomputer system in civilian use at the time

private vs public sequencing

what was the scandal with the HGP?

both were basically shit talking each other; Celera DNA source was originally reported as 20 anonymous donors'; Later revealed that the Celera genome mostly represents Craig Venter (company founder)

main benefit of HGP

Improved the practice of medicine (diagnostics, prognostics, management, treatment); made genomic medicine possible

cost of mapping human genome went from ___ to ___

100 million to 500

size of human genome

3.1 Gb

how many different DNA molecules in XX vs XY

23 and 24

how many protein-coding genes

unsure but estimated to 20,000

percentage of protein-coding genes

1-2%

number of RNA genes

uncertain, perchance 20,000

gene density of human genome

1/80kb

number of psuedo genes

15,000

percentage of genome that is repetive

50-67%

what is spliced during RNA processing?

introns

what codes for protein

exon

is promoter considered part of the gene?

no! :p

___ and __ represent the largest protein-coding genes (nucleotide content)

CNTNAP2 and DMD

mean gene size

64.9 kb

mean exon size

268 bp

how many exons per gene?

average of 9

larger genes typically have larger ___

introns

because of how much energy it takes to splice introns out, largely expressed genes typically have ___

short to no introns

around ___ protein-coding genes are associated with a disease so far

5000

something can be a longer gene but ends up translating into a shorter protein, why?

lots of introns bc gene is highly regulated

how does sequencing detect the protein-coding gene?

look for ORF with start and stop codon; often conserved evolutionarily so similar size can help us determine if it is a real gene

Gene number found to be _____ when genome sequence of other mammals was obtained (mouse and dog); gene counterparts in other mammals failed to be found

inflated

what is readthrough transcription

conjoined genes, when a smaller gene is embedded in introns of another gene

genes are often high density in which part of the chromosome?

subtelomeric spaces

if 1-2% of genome is protein-coding genes, what is the remainder?

highly repetitive DNA

satellite DNA

high copy number tandemly repeated DNA (heterochromatin)

ranges for satellite, mini and micro satellite

Satellite DNA (>100 kb), Minisatellite DNA (100 bp - 20 kb), Microsatellite DNA (<100 bp)

what is function of satellite dna

contributes to formation of crucial chromosome structure, heterochromatin establishment, genome stability, and development

what are transposon repeats

DNA that can change its position; increase genetic diversity and can also regulate gene expression; May induce human genetic disease (insertional mutagenesis)

what are the two mechanisms by which genes jump around?

retrotransposons and dna transposons

transposase

cuts the transposon part of gene and relocates it to new location (cut and paste mechanism)

explain retrotransposon

they transcribe DNA into an RNA intermediate and then retrotranscribe into DNA in a different location in DNA

what are the three types of retrotransposons

LINES: transcribe their own RNA, convert it back into DNA, and insert it elsewhere without needing help from other elements.

SINES: "hijack" the reverse transcriptase and endonuclease enzymes produced by nearby LINEs to copy and insert themselves

retrovirus-like elements: persist within the human genome as remnants of ancient retroviral infections (code reverse transcriptase and integrase)

how do dna transposon repeats work

cut and paste mechanism using transposase

what does LINEs stand for

long interspered retrotransposable elements

what does SINEs stand for

short interspersed retrotransposable elemtns

how can transposon negatively impact an individual and give an example

the dna fragment can insert itself in the middle of another gene and disrupt its transcription

ie. apc gene is a tumor suppressor and if a transposon inserts itself, it will no longer suppress cancer

when did whole genome duplication last happen

hundreds of millions of years ago but not many were conserved evolutionarily

tandem duplication

unequal crossing over of homologoous chromosomes and sister chromatids

duplicative transposition by recombination

unstable regions of euchromatin recombine with other chromosomes

rna-directed duplicative transposition

RNAs are reversed transcribed into cDNA and integrated into a new chromosomal location

types of active duplication

rna-directed duplicative transposition, duplicative transposition by recombination, tandem duplication, whole genome duplication

psuedogenes

nonfunctional segments of DNA that resemble functional genes but have been mutated into an inactive form over the course of evolution

three types of puedogenes

non-processed, processed, unitary

what is non-processed psuedogene and what percentage of genome

whole genome duplication in which one gene copy has become inactive due to mutation, 25%

what is processed psuedogene and what percentage of genome

copies of mRNAs that are reverse transcribed into cDNA and integrated into the genome; lack a promoter, and have usually accumulated mutations, 75%

unitary psuedogene

formerly functional gene is no longer required (not evolutionarily advantageous) and is inactivated by mutation (no functional counterpart in the same genome)

what is the largest gene superfamily in the human genome

olfactory receptor proteins

how can psuedogenes cause disease

Non-allelic Homologous Recombination, since the psuedogene can seem similar to regular gene, cell can get confused

why are psuedogenes hard to test diagnostically

Because the sequence of the active gene and the inactive pseudogene are nearly identical, PCR primers often accidentally bind to and amplify both at the same time.

types of small variants (<50bp)

snp, insertion, deletion, indel

types of structural variants (> 50bp)

copy number variant, inversion, translocation

On average, each child is born with ____ new sequence variants (de novo)

around 70

segmental duplication

Segmental duplications are blocks of DNA that typically share 90% sequence identity (~5% of the euchromatin; present only within higher order primates so suggests recent evolutionary origin

why is segmental duplication important in human genetics

many deletion/duplication-related disorders result from recombination between them

copy number variants are adaptive, give an example

populations that consume high-starch diets have more copies of the gene that codes for salivary amylase

how does dna fingerprinting work

Assesses repetitive regions of genomic DNA (mini/microsatellite DNA); unique signature; first conducted using souther blot but now uses pcr amplifying specific short tandem repeats across genome

how does southern blot method of dna fingerprinting work

1. Isolate DNA from blood sample

2. Digest DNA with restriction enzyme(s)

3. Separate DNA fragments using gel electrophoresis

4. Transfer DNA fragments to a nitrocellulose membrane

5. Incubate membrane with radioactively labeled DNA probes (target satellite DNA)

6. Expose to film to visualize labeled DNA fragments

how does the pcr method of dna fingerprinting work

1. Isolate DNA from blood sample

2. PCR amplify STRs of interest

3. Separate DNA fragments using gel electrophoresis

4. Stain DNA fragments (e.g. EtBr)

5. Visualize DNA fragments using UV light source

______ sequencing is still gold standard for sequencing, but newer technologies are more accurate

sanger

sanger vs next gen: gel electrophoresis

needed vs not needed

sanger vs next gen: automation of sequencing

semi automated vs fully automated

sanger vs next gen: recording of sequence during seuqnecing reaction

not possible vs possible

sanger vs next gen: dna template prep

cloned in cells and amplified by PCR vs fragments ligated and amplified by PCR

sanger vs next gen: read lengths

up to 800 nucleotides vs up to 14000 nucleotides

sanger vs next gen: number of DNA templates sequenced per reaction

one vs millions

sanger vs next gen: sequencing error rates

very low vs high for individual fragments but comparing multiple reads is low

sanger vs next gen: applications

routine small scale vs global analyses

illumina sequencing

short read sequencing (typcially 150 bp in length), millions of fragments simultaneously; sequence determined during DNA synthesis

how has illumina sequencing improved

went from 13 years for 1 genome (using Sanger) to 20000 genomes in 1 year