HUGE Lecture 1 (08/25)

How many pairs of chromosomes do humans have?

23 pairs

How many pairs of autosomes do humans have

22

What are the 2 sex chromosomes?

XX- female
XY- male

How many total chromosomes are in the human nucleus

46

How are autosomes numbered

Largest to smallest

How is DNA packaged

DNA is tightly wound around DNA associated proteins called histones

What are chromatins

histones + DNA tightly wound

Types of chromatin

euchromatin and heterochromatin

What is euchromatin

Open state of chromatin that is transcriptionally active for RNA polymerase to transcribe DNA

What happens to chromatin during interphase

Decondenses (“opens”) and becomes transcriptionally active

What type of chromatin is most of the human genome

euchromatin

Gene density of euchromatin

high

What is heterochromatin

tightly packed DNA that remains condensed throughout the cell cycle, mostly transcriptionally inactive

Gene density of heterochromatin

low

DNA sequence feature of heterochromatin

many repetetive DNA sequences

Roll of heterochromatin in repetitive DNA

maintains genome stability by silencing repetitive DNA

Roll of heterochromatin in cell identity

represses genes to preserve cell identity

Roll of heterochromatin in organization of chromosomes

ensures the proper organization of chromosomes during cell division and within the nucleus

Two reasons why euchromatin the initial focus of human DNA sequencing

1. Euchromatin is where most genes are located

2. Heterochromatin contains a lot of repetitive DNA sequences which are difficult to sequence

Goals of human genome project

sequence entire human genome, identify genes, understand gene function in the context of disease

How many years and how much money was put into the human genome project

13 years (1990-2003) and 3 billion dollars

What were the main impacts of the human genome project

• advancing policies

• increased support for open sharing of scientific data

• greater emphasis on biomedical ethics

What did the human genome project help develop technologically

improved mapping and sequencing technologies

What other model organisms’ genomes were sequenced

e coli, baker’s yeast, fruit fly, nematode, and mouse

What was the opposition to the human genome project

Whether the cost and time was worth what we would learn and any resulting healthcare improvements.

What sequencing technique did the Human Genome Project (HGP) use to sequence the human genome

Sanger sequencing

Who invented Sanger sequencing

Frederick Sanger

What was Sanger awarded the Nobel Prize for

Determining the first protein sequence (insulin) and inventing Sanger sequencing

What copies DNA from a template strand

DNA polymerase synthesizes DNA from a template strand in the 5’ to 3’ direction

How are new nucleotides added to the growing DNA chain

DNA polymerase links the alpha phosphate of the incoming deoxyribonucleoside triphosphate to the 3’ hydroxyl group at the 3’ end of the growing chain.

What does addition of dideoxynucleotide triphosphates do to chain elongation by DNA polymerase

dideoxynucleotide triphosphates arrest chain elongation since there is no 3’ hydroxyl group to form a phosphodiester bond

How does Sanger sequencing work

• Primers and a DNA polymerase are used to make copies of DNA sequences of interest

• A single oligonucleotide primer is used to make labeled DNA copied of desired sequence

• DNA synthesis continues when dNTPs are used but ddNTP incorporation immediately terminates synthesis

• Fluorescent dyes label ddNTPs and also their corresponding DNA fragments

• DNA fragments are separated by size using gel electrophoresis

• Lasers are used to excited DNA-labeled fluorophores and fluorescent signals are recorded

Sanger sequencing pitfalls

• limited throughput- one DNA fragment sequenced at a time

• not cost effective

• requires a large amount of template DNA to produce clear results

What is the max and average fragment length that can be sequenced

max is ~850 bp, averages between 400-600 bp for a clean read

What were the two competing approaches to sequencing the human genome

• publicly funded HGP- cloned DNA fragments then mapped to determine sequence order

• private company Celera- shotgun sequencing

Human Genome Project (HGP) approach to sequencing the human genome

• Lyse human cells

• Extract DNA from nucleus

• Partial digestion with restriction endonuclease

• Join DNA to vector molecules and clone in bacterial or yeast cells

• Sequence cloned DNA

• Assemble sequenced clones based on overlapping base content

What is a clone contig

amalgamations of several sequenced fragments that can be pieced together to make up a full chromosome

Celera approach to sequencing the human genome

• Fragment large amounts of genomic DNA

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

Issue with Celera’s approach

assembling the large number of DNA fragments was difficult and required them to build the most powerful supercomputer system in civilian use at the time

Would Celera have been able to assemble the human genome without the HGP

No, they also had access to the open data the HGP published at the end of each day and used it to train their algorithm

Timeline of the “complete” genome

• 2001- both “complete” genomes published, but they were still drafts

• 2003- HGP produced a 90% complete human genome sequence- just euchromatin

• 2022- telomere to otelomere consortion published the first complete genome with no gaps in the assembly

Benefits of HGP

• improved genetic understanding of disease and assessment of human disease risk

• made genomic medicine possible

• resulted in newer sequencing technologies

Unintended consequences of HGP

Narrows the range of variation that can be considered “normal”- could devalue human diversity if used improperly

Is Sanger sequencing still used

It is but mostly used for confirmation of other sequencing analyses