One cell is all you need

ORIGINAL: News Harvard

By Peter Reuell. Harvard Staff Writer

January 4, 2013

Innovative technique can sequence entire genome from single cell

Rose Lincoln/Harvard Staff Photographer. Mallinckrodt Professor of Chemistry and Chemical Biology Xiaoliang Sunney Xie (from left) has co-authored a paper on gene sequencing with graduate student Alec Chapman and postdoctoral fellow Chenghang Zong. The paper demonstrates a new method for DNA amplification that could signal a breakthrough in genomics.

The notion that police can identify a suspect based on the tiniest drop of blood or trace of tissue has long been a staple of TV dramas, but scientists at Harvard have taken the idea a step further. Using just a single human cell, they can reproduce an individual’s entire genome.

As described in a Dec. 21 paper in Science, a team of researchers, led byXiaoliang Sunney Xie, the Mallinckrodt Professor of Chemistry and Chemical Biology, and made up of postdoctoral fellow Chenghang Zong, graduate student Alec Chapman, and former graduate student Sijia Lu, developed a method — dubbed MALBAC, short for Multiple Annealing and Looping-based Amplification Cycles — that requires just one cell to reproduce an entire DNA molecule.

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More than three years in the making, the breakthrough technique offers the potential for early cancer treatment by allowing doctors to obtain a genetic “fingerprint” of a person’s cancer from circulating tumor cells. It also could lead to safer prenatal testing for a host of genetic diseases.

“If you give us a single human cell, we report to you 93 percent of the genome that contains three billion base pairs, and if there is a single base mutation, we can identify it with 70 percent detectability, with no false positives detected,” Xie said. “This is a major development.”

In a second paper, published simultaneously, researchers from Xie’s lab worked with scientists at Peking University in China to demonstrate MALBAC by sequencing 99 sperm cells from one individual and examining the paternal and maternal contribution to each cell’s genome.

As its name suggests, Xie said, MALBAC is a type of DNA amplification that allows researchers to duplicate the single DNA molecule present in a cell many times so it can be analyzed in the lab.

“While other methods of DNA amplification exist, most — like polymerase chain reaction (PCR) or multiple displacement amplification (MDA) — suffer from a specific problem,” Xie said. “Because they amplify exponentially, both have bias. They dramatically amplify some parts of the genome, but amplify others very little.”

By comparison, he said, MALBAC relies on linear amplification, meaning it is able to minimize the sequence-dependent bias.

Just as it does with other methods, the amplification process begins by splitting the DNA double helix into two single strands. Xie’s team then adds a random “primer” — tiny fragments of DNA — that binds in dozens of locations along each strand.

To extend those primers, Xie’s team used a DNA polymerase, the same cellular “machine” that synthesizes DNA as cells divide. Using that machine, researchers are able to extend the primers from as few as seven bases to as many as 2,000. Upon heating, they break the elongated primers apart from the original DNA, yielding half products.

When those half products are then amplified using the same primers, the two ends of the DNA combine, forming a loop that prevents it from being amplified again. The leftover half products and the original DNA are subject to another cycle of amplification. After five cycles of such linear pre-amplification, the full product is amplified by PCR to produce enough material for sequencing.

Despite the high coverage, DNA polymerases do occasionally make errors, Xie explained. To ensure that the genome produced by MALBAC is accurate, researchers turned to a different technique.

“Many diseases are associated with a single base mutation,” Xie said. “The challenge, however, is that finding one mutation in more than 3 billion base pairs is like looking for a needle in a haystack. Earlier techniques, like PCR or MDA, start with many cells, making the challenge even greater; a single mutation simply gets lost in the process. MALBAC, however, starts with a single cell, so it is easier to identify those mutations when they happen.”

To ensure MALBAC’s accuracy, Xie’s team simply let the original cell divide.

While the polymerase that researchers use to build the DNA sequence is highly accurate, only making one mistake per 10,000 bases, letting the cell divide gives researchers a chance to double check its work.

“The chances of the same mistake being made at the same base position are about one in 100 million,” Xie said. “If we let the cells divide again, and sequence three cells, the chances go up to one in 10 billion, less than the number of bases in the entire DNA molecule, so we can remove all the false positives.

“Getting that level of accuracy is very important, because if a doctor tells a patient that he detects a mutation, he doesn’t want to be wrong,” he continued. “When we use MALBAC, if a mutation appears in two or three related cells, we know it must be a real mutation.”

As a demonstration of MALBAC’s power, Xie and his team monitored the mutations that arose in a single cancer cell as it divided over 20 generations, and uncovered as many as 50 newly acquired mutations.

“This is the first time the mutation rate of a human cell has been measured directly,” Xie said. “Because we can now see the unique, newly acquired bases, we can study the dynamics of the genome in a way that was not possible before.”

Ancient Incan Mummy Had Lung Infection, According to Novel Proteomics Analysis

ORIGINAL: ScienceDaily

ScienceDaily (July 25, 2012) — A 500-year-old frozen Incan mummy suffered from a bacterial lung infection at the time of its death, as revealed by a novel proteomics method that shows evidence of an active pathogenic infection in an ancient sample for the first time. 

The full report is published July 25 in the open access journal PLoS ONE.

Detecting diseases in ancient remains is often fraught with difficulty, especially because of contamination. Techniques based on microbe DNA can easily be confused by environmental contamination, and they can only confirm that the pathogen was present, not that the person was infected, but the researchers behind the study, led by Angelique Corthals of the John Jay College of Criminal Justice, City University of New York, found a way around this problem. They used proteomics, focusing on protein rather than DNA remains, to profile immune system response from degraded samples taken from 500 year-old mummies.

The team swabbed the lips of two Andean Inca mummies, buried at 22,000-feet elevation and originally discovered in 1999, and compared the proteins they found to large databases of the human genome. They found that the protein profile from the mummy of a 15-year old girl, called "The Maiden," was similar to that of chronic respiratory infection patients, and the analysis of the DNA showed the presence of probably pathogenic bacteria in the genus Mycobacterium, responsible for upper respiratory tract infections and tuberculosis. In addition, X-rays of the lungs of the Maiden showed signs of lung infection at the time of death. Proteomics, DNA, and x-rays from another mummy found together with the Maiden did not show signs of respiratory infection.

"Pathogen detection in ancient tissues isn't new, but until now it's been impossible to say whether the infectious agent was latent or active," says Corthals. "Our technique opens a new door to solving some of history's biggest mysteries, such as the reasons why the flu of 1918 was so devastating. It will also enhance our understanding of our future's greatest threats, such as the emergence of new infectious agents or re-emergence of known infectious diseases."

"Our study is the first of its kind since rather than looking for the pathogen, which is notoriously difficult to do in historical samples, we are looking at the immune system protein profile of the "patient," which more accurately tells us that there was indeed an infection at the time of death." or "Our study opens the door to solving many historical and current biomedical and forensic mysteries, from understanding why the plague of 1918 was so lethal, to finding out which pathogen is responsible for death in cases of multiple infections."