DNA World: 2018

Transformation of DNA

Remove a trace of E. coli cells from the glycerol stock vial with a sterile toothpick or inoculating loop, and streak it out on LB-agar plates containing an appropriate concentration of the relevant selective antibiotic(s) (see Antibiotics). If the host strain has already been cultured and stored at 2–8°C (cultures can be stored at 2–8°C for up to 3 months without any significant loss of viability), streak out bacteria from those stocks.
Incubate at 37°C overnight.
Pick a single colony and inoculate 10 ml LB medium containing relevant antibiotic(s). Grow overnight at 37°C.
Add 1 ml overnight culture to 100 ml prewarmed LB medium containing the relevant antibiotic(s) in a 500 ml flask, and shake at 37°C until an OD₆₀₀ of 0.5 is reached (approximately 90–120 min).
Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottom centrifuge tube.
Collect the cells by centrifugation at low speed (5 min, 4000 x g, 4°C).
Discard the supernatant carefully. Always keep the cells on ice.
Resuspend the cells gently in cold (4°C) TFB1 buffer (30 ml for a 100 ml culture) and keep the suspension on ice for an additional 90 min.
Collect the cells by centrifugation (5 min, 4000 x g, 4°C).
Discard the supernatant carefully. Always keep the cells on ice.
Resuspend the cells carefully in 4 ml ice-cold TFB2 buffer.
Prepare aliquots of 100–200 µl in sterile microcentrifuge tubes and freeze in liquid nitrogen or a dry-ice–ethanol mix. Store the competent cells at –70°C.

A quick DNA purification protocol

A quick "dirty" prep is usually sufficient, while some genotyping may work better with highly purified DNA. Determine empirically which protocol works best for your genotyping.

1. NaOH extraction (quick "dirty" DNA preparation). Reference: Truett GE et al. 2000. Biotechniques 29(1):52-54

Cut 2mm of tail and place into an Eppendorf tube or 96-well plate.
Add 75ul 25mM NaOH / 0.2 mM EDTA.
Place in thermocycler at 98ºC for 1 hour, then reduce the temperature to 15°C until ready to proceed to the next step.
Add 75ul of 40 mM Tris HCl (pH 5.5).
Centrifuge at 4000rpm for 3 minutes.
Take an aliquot for PCR (use 2 ul undiluted, or 2 ul of a 1:100 dilution/reaction).

DNA Isolation from Blood Samples

1. Obtain 65-100 µl of blood by retro-orbital bleed with a heparinized microcapillary tube. Expel blood immediately into a 1.5 ml microfuge tube containing 20 µl of 10 mM EDTA. Mix immediately to prevent clot formation. Store on ice until processing.

2. Add 200 µl lysis buffer to each tube and vortex to suspend evenly.

3. Microfuge 25 seconds at 16000xg to pellet nuclei.

4. Remove and discard supernatant and repeat steps 2-4 two more times, or until no hemoglobin remains.

5. Resuspend nuclear pellet in 100 µl PBND with 60 µg/ml proteinase K and incubate at 55 C for 60 minutes (or overnight, if convenient).

6. Heat samples to 97 C for 10 minutes to inactivate proteinase K.

7. Add 1-5 µl of DNA solution for a 25 µl PCR reaction.

Reagents:

1) Lysis buffer

0.32 M sucrose
10mM Tris-HCl (pH 7.5)
5 mM MgCl2
1% v/v Triton X-100

2) PBND (PCR buffer with nonionic detergents)*

50 mM KCl
10 mM Tris-HCl (pH 8.3)
2.5 mM MgCl2
0.1 mg/ml gelatin
0.45% (v/v) Nonidet P40
0.45% (v/v) Tween 20
Autoclave to sterilize and dissolve gelatin.
Store frozen.

*Add proteinase K (60 µg/ml) immediately prior to use).

(Adapted from Higuchi, R. (1989) Amplifications 2: 1-3)

Pakistan Zainab murder: DNA suggests suspect in other case was 'innocent'

Pakistani officials say they will investigate allegations police carried out an unauthorised killing of a man wrongly accused of murdering a child.

Police shot Mudasir, accused of killing five-year-old Iman Fatima, in February 2017, saying he tried to escape.

However, a BBC Newsnight investigation has uncovered allegations that Iman's real killer is still at large.

DNA tests suggest Iman was killed by the same man suspected of murdering another child this month.

The rape and murder of six-year-old Zainab Ansari earlier this month sparked outrage and protests across Pakistan.

Police working on the investigation into Zainab's murder have discovered DNA traces matching those found in seven other attacks on young girls in the same city.

Out of the seven attack victims, four - including Iman - were murdered.

Until now, police and Iman's family thought her case had already been solved - but her family now believe the real killer is still on the run, while Mudasir was innocent.

'Trying to escape'?

Iman Fatima had been playing outside in the street with her five-year-old cousin Adeel.

Sitting next to his father, Adeel told the BBC, "The kidnapper made me stand against the wall and took Iman Fatima away. He took her upstairs, put her in a sack and took her away."

Adeel's memory of the incident is at times vague and confused but his family say after the abduction, despite his age, he identified the house his cousin had been taken into, and later the man responsible.

The suspect was 21 year-old Mudasir, a factory worker who had moved to Kasur with his family around two years earlier.

What happened next is disputed. According to one police version Mudasir was killed resisting arrest. Another senior police officer told the BBC he was taken into custody where he confessed, and then was killed "trying to escape."

But in an exclusive interview with the BBC, Mudasir's family said they believed police killed him despite knowing he wasn't the killer because they were not able to find the real culprit.

Human rights groups have criticised police in Pakistan for carrying out what are known locally as "police encounters" - extra judicial killings that are then covered up as incidents where suspects resisted arrest.

Analysts say police often carry out "encounter killings" because of the low conviction rate in Pakistani courts.

'They killed my son'

After the discovery of Iman Fatima's body local residents began to protest against the perceived inaction by authorities. A month earlier in January 2017, another 5-year-old girl, Ayesha Asif had also been sexually assaulted and murdered.

Mudasir's mother Jamila Bibi told the BBC, "I feel as if I have lost everything. They killed my son."

The family moved out of Kasur just days afterwards, "No-one in the neighbourhood would even talk to us" Jamila Bibi added tearfully.

Mudasir's family say he was taken into custody the same evening Iman Fatima was abducted and killed. They allege they then went with police to recover his body from a local hospital.

Police officials in Kasur told the BBC that Mudasir had been positively identified as the suspect by the girl's cousin Adeel.

Speaking to the BBC, a relative of Iman Fatima said police had called him into the police station to hear Mudasir confess to the killing.

But the DNA evidence examined as part of the enquiry into Zainab's murder later suggested Mudasir was not the killer.

Following Mudasir's death in February 2017, another four young girls were attacked, including Zainab. Three of them died, one is still in hospital. Traces of the same DNA were found on their bodies as of Iman Fatima.

When presented with the allegations uncovered by the BBC, Malik Ahmad Khan, spokesman for the Punjab Government, told the BBC that authorities in the province would carry out a "full-fledged inquiry" and that those responsible for any "extrajudicial killing won't be spared."

Iman Fatima's father told the BBC, "I'm so worried - the real killer is still roaming free, and an innocent man has been killed."

"I am so angry with the police I can't explain. We want justice and we want the actual culprit caught."

Ancient DNA tests solve Manchester mummy sibling mystery

A pair of Egyptian mummies nicknamed "Two brothers" by a museum are actually half-brothers, according to research using ancient DNA.

Scientists from the University of Manchester used DNA from the mummies' teeth to establish that they have the same mother but different fathers.

"I am sure the brothers would be very proud of us," said Dr Konstantina Drosou, from the university.

The pair are Manchester Museum's oldest mummies and date back to 1800BC.

Egyptologists have long debated whether the mummies of two elite men - Khnum-nakht and Nakht-ankh - were related ever since they were discovered in 1907.

A university spokesman said two years ago "ancient DNA" was extracted from their teeth to solve the mystery.

Dr Konstantina Drosou, from the university's School of Earth and Environmental Sciences, tested the samples using DNA sequencing - the process used to determine the order of the four chemical building blocks that make up the genetic information from a given living organism.

"It was a long and exhausting journey to the results but we are finally here," she said.

"I am very grateful we were able to add a small but very important piece to the big history puzzle and I am sure the brothers would be very proud of us. These moments are what make us believe in ancient DNA."

The pair's joint burial site, later called The Tomb of The Two Brothers, was discovered at Deir Rifeh, a village 250 miles (400km) south of Cairo, by Egyptian workmen directed by early 20th Century Egyptologists, Flinders Petrie and Ernest Mackay.

Hieroglyphic inscriptions on the coffins indicated that both men had mothers with the same name, Khnum-aa.

Alcohol may cause cancer by damaging stem cell DNA

Drinking alcohol may cause irreversible damage to the DNA of stem cells and increase the risk of developing certain cancers, a new study has revealed.

Using genetically modified mice, British scientists found that acetaldehyde, a toxin produced as the body processes alcohol, can slice through the DNA within blood stem cells and cause permanent damage.

In the study, published recently in the journal Nature, the mice lost their ability to reproduce fresh blood after being given diluted alcohol for ten days.

Sequencing the genome of the stem cells indicated that their DNA had been scrambled to the point that the cells no longer functioned.

When healthy stem cells are faulty, they can give rise to cancerous cells, particularly in the mouth, throat, oesophagus, breast and so on.

The effects of such damage can be neutralized, however, by the body's two-layer defence mechanism. The first layer is a protective enzyme called aldehyde dehydrogenase 2 (ALDH2), which prevents build-ups of acetaldehyde, and the second repairs the DNA damage.

In the study, mice lacking ALDH2 suffered four times as damage compared with the other ones.

About 8 percent of the world's population, mostly those of East Asian ancestry, are born with an deficiency in ALDH2.

"Our study highlights that not being able to process alcohol effectively can lead to an even higher risk of alcohol-related DNA damage and therefore certain cancers," said Ketan Patel, who led the research at the MRC Laboratory of Molecular Biology in Cambridge.

DNA-based compound may reduce damages of Alzheimer's disease: study

Researchers at Washington University School of Medicine in St. Louis have created a kind of DNA-base molecule targeting APOE protein. APOE is a major Alzheimer's risk gene.

The study has been published on Dec. 6 in the journal Neuron.

The molecule, known as an antisense oligonucleotide, interferes with the instructions for building the APOE protein.

The researchers injected the compound into the fluid surrounding the brains of newborn mice. For comparison, they gave other newborn mice either saltwater or a placebo "oligo" that does not interfere with the APOE instructions. Levels of APOE protein dropped by about half in mice given the APOE compound as compared with those that received the placebo oligo or saltwater.

Two months later, the researchers gave the mice a booster dose of the treatment or the saltwater. They examined the mice's brains at 4 months old. The mice that received the APOE antisense oligos had about half as many amyloid plaques as mice given saltwater. Each plaque triggered only half as much damage to nearby neurons, an indicator that the compound had prevented some of the neurological damage that leads to Alzheimer' s disease.

The researchers then tested whether giving the APOE compound after amyloid plaques appear could prevent further changes to the brain. They introduced either the APOE compound or saltwater into the fluid surrounding the brains of 6-week old mice, and then examined the mice's brains at 4 months old. They found no difference in the number of plaques or the total amount of amyloid beta between the mice that received the compound and those given just saltwater. The compound failed to reduce the amount of amyloid in the mice's brains.

However, in the mice treated with the APOE compound, each plaque triggered only about half as much damage to the surrounding neurons. This suggests that even a late start could reduce the harm posed by amyloid beta.

"If you wanted to target APOE to affect the amyloid process, the best thing would be to start before the plaques form," said senior author David Holtzman, professor and head of the Department of Neurology of Washington University at St. Louis. "But even if you start later, you still may reduce the amount of damage caused by the plaques."

Alzheimer's, which affects one in 10 people over age 65, is marked by brain plaques made of a sticky protein known as amyloid beta. The plaques start forming in the brains of Alzheimer's patients years before the characteristic symptoms of memory loss and confusion appear. APOE4 raises the risk of Alzheimer's partly by encouraging amyloid beta to collect into damaging plaques. People with APOE4 face up to 12 times the risk of developing Alzheimer's than the general population.

"Scientists have been interested in APOE for years but there are only a few examples where researchers have targeted it with a compound in living animals," said Holtzman. "Our findings indicate that APOE is not just involved in Alzheimer's risk and disease progression, but it could potentially be a real target for treatment or prevention."

DNA lab for identifying missing persons opens in The Hague

The International Commission on Missing Persons (ICMP) on Tuesday opened a state-of-the-art DNA laboratory at its new headquarters in The Hague.

The laboratory system focuses exclusively on missing persons identification and utilizes Next Generation Sequencing (NGS), a technique that is expected to deliver an exponential increase in the power of DNA identification, according to ICMP, a treaty-based international organization with a mandate to secure the cooperation of governments and others in locating and identifying missing persons.

Since 2001, ICMP has operated a DNA laboratory system to assist governments in identifying large numbers of persons missing from conflict, human rights abuses, natural and manmade disasters, organized crime, irregular migration and other circumstances where persons go missing for involuntary reasons.

The system, which is designed to work on the most challenging cases, operates on a high-throughput scale. It has generated DNA results on more than 50,000 cases of degraded skeletal remains and contributed to the identification of around 20,000 persons worldwide.

With the new laboratory, ICMP aspires to maintain a capacity of up to 10,000 cases a year.

Over the course of two decades, ICMP has responded to a wide variety of complex missing persons scenarios in over 40 countries.

Scientists create DNA robots for future delivering drugs in human body

Scientists have created miniature robots out of DNA that can autonomously "walk" around a surface, pick up certain molecules and drop them off in designated locations, a new study published Thursday in the U.S. journal Science said.

"Just like electromechanical robots are sent off to faraway places, like Mars, we would like to send molecular robots to minuscule places where humans can't go, such as the bloodstream," said LuLu Qian, assistant professor of bioengineering of the California Institute of Technology.

Such technology could one day be used for a wide range of applications, including synthesizing therapeutic chemicals in an artificial molecular factory, delivering drugs in bloodstreams or cells, or sorting molecular components in trash for recycling, Qian said.

To create a DNA robot, Qian's team constructed three basic building blocks, including a "leg" with two "feet" for walking, an "arm" and "hand" for picking up cargo, and a segment that can recognize a specific drop-off point and signal to the hand to release its cargo.

Each of these components is made of just a few nucleotides within a single strand of DNA.

In principle, these modular building blocks could be assembled in many different ways to complete different tasks.

For example, a DNA robot with several hands and arms, could be used to carry multiple molecules simultaneously.

In the work described in the Science paper, the Qian group built a robot that could explore a molecular surface, pick up two different molecules and then distribute them to two distinct regions on the surface.

"The DNA robot moves around on a 58-nanometer-by-58-nanometer pegboard on which the pegs are made of single strands of DNA complementary to the robot's leg and foot," Qian's team said in a statement.

"The robot binds to a peg with its leg and one of its feet -- the other foot floats freely," the team said.

"When random molecular fluctuations cause this free foot to encounter a nearby peg, it pulls the robot to the new peg and its other foot is freed. This process continues with the robot moving in a random direction at each step."

Since one step for the little guy takes five minutes, and allows it to move six nanometers, it may take a day for the robot to explore the entire board, the team said.

"Along the way, as the robot encounters cargo molecules tethered to pegs, it grabs them with its 'hand' components and carries them around until it detects the signal of the drop-off point," it said.

"The process is slow, but it allows for a very simple robot design that utilizes very little chemical energy."

In a summary on the robot, the Science magazine called Qian's work "one small step for a DNA robot, one giant leap for mankind."

"The future is here," the summary wrote.

Ancient Eurasian DNA sequencing reveals how modern humans migrate

Chinese researchers are discovering unexpected genetic connections between individuals on opposing sides of Eurasia with direct DNA sequencing.

Their study suggests a complex history that may represent early gene flow across Eurasia or an early population structure that eventually led to Europeans and Asians.

In a review published in the journal Trends in Genetics on Thursday, scientists at the Chinese Academy of Sciences in Beijing discuss the genetics of ancient individuals from Europe and Western Asia between 45,000 to 7,500 years ago.

The authors summarized work that investigated the genomes of more than 70 ancients in the Eurasian family tree.

"Aside from these individuals, it is a fact that sampling for the Eurasian region is sparse for all time periods except the present-day," says the correspondent author Fu Qiaomei, a paleogeneticist at the Chinese Academy of Sciences.

"But with the information from the several individuals available for ancient DNA sequencing we do have hints at interesting population structure, migration and interaction in East Asia."

The researchers learned that in Eurasia between 35,000 and 45,000 years ago, at least four distinct populations were present. These were early Asian and Europeans, as well as populations with ancestry hardly found or not at all in modern populations.

It shows that, by 7,500 to 14,000 years ago, the populations across Eurasia shared genetic similarities, suggesting greater interactions between geographically distant populations.

These analyses also revealed at least two Neanderthal population mixing events, one approximately 50,000 to 60,000 years ago and a second more than 37,000 years ago.

Fu and colleagues hope to extend this type of sequencing and analysis to learn more about the genetic prehistory of East Asia and other regions, including Oceania, Africa, and the Americas.

"All of those areas have a rich human prehistory, particularly in Africa, so any ancient DNA from those continents will likely resolve some major questions on human migration," she says.

DNA Modification/Epigenetics

Analysis of Mitotic Checkpoint Function in Xenopus Egg Extracts

Yinghui Mao

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot099853

Abstract Full Text (PDF)

Analysis of DNA Methylation in Mammalian Cells

Paul M. Lizardi, Qin Yan, and Narendra Wajapeyee

Cold Spring Harb Protoc 2017; doi:10.1101/pdb.top094821

Jan 27, 2018

'Trying to escape'?

'They killed my son'