What is the method of separating molecules such as DNA according to size?

Gel electrophoresis is used to separate macromolecules like DNA, RNA and proteins. DNA fragments are separated according to their size. Proteins can be separated according to their size and their charge (different proteins have different charges).

Table of Contents Show

When is gel electrophoresis used to separate DNA fragments?
When is gel electrophoresis used to separate proteins?
Size Separation
Charge and pH separation
Two-dimensional electrophoresis
DNA profiling
How is a DNA profile produced today?
Solving crime
How are DNA profiles stored?
Linking blood relatives

A solution of DNA molecules is placed in a gel. Because each DNA molecule is negatively charged, it can be pulled through the gel by an electric field. Small DNA molecules move more quickly through the gel than larger DNA molecules.

The result is a series of ‘bands’, with each band containing DNA molecules of a particular size. The bands furthest from the start of the gel contain the smallest fragments of DNA. The bands closest to the start of the gel contain the largest DNA fragments.

When is gel electrophoresis used to separate DNA fragments?

Gel electrophoresis can be used for a range of purposes, for example:

When is gel electrophoresis used to separate proteins?

Thanks to TV shows like CSI, many people are familiar with the use of gel electrophoresis to separate macromolecules like DNA. However, gel electrophoresis can also be used to separate out proteins.

Different proteins have different sizes, mainly due to the number of amino acid building blocks in their structure. Chemical modifications attached to the protein also affect its size. Different proteins also have different charges. This can result from both the types of amino acid used to construct them, as well as the types of modifications attached to them.

Different types of electrophoresis gels are used to provide different types of information. The type of gel you choose therefore depends on the type of question you are asking.

Size Separation

Typically, gels made from polyacrylamide are used to separate proteins on the basis their different sizes. Usually, the proteins are first treated with heat and a chemical called SDS in order to unravel the protein. SDS is a detergent that gives all the proteins the same overall negative charge so that when an electric current is applied to the gel, separation is only due to the size of the protein. This technique is called SDS-PAGE (SDS-Polyacrylamide gel electrophoresis).

Small protein molecules move more quickly through the gel than larger proteins, resulting in a series of ‘bands’. Each band contains a protein of a particular size. These can be compared with standards of known sizes.

An SDS-PAGE gel has been used to separate proteins on the basis of size. The samples are the blood of various shark species. The first lane contains markers of known sizes. Large proteins are at the top of the gel and small proteins are at the bottom.

This technique might be used for many purposes, including purifying a particular protein, for example to isolate an enzyme for the food industry.

Charge and pH separation

Isoelectric focusing (IEF) and agarose gel electrophoresis are two ways that proteins can be separated by their different electrical charges. Unlike SDS-PAGE, the proteins are usually kept in their native (folded) state. The type of gel that is used, and the solution around the gel, are also different.

In agarose gel electrophoresis, proteins are loaded in the middle of the well. Those with a strong negative charge move fastest towards the positive side of the gel, whereas positively charged proteins move in the opposite direction.

This technique might be used to separate proteins that have the same molecular weight but different charges, or when size is not important (e.g. to look at changes in the presence of different protein during the development of a disease).

Two-dimensional electrophoresis

These days, charge (IEF) and size (SDS-PAGE) separation are often employed together in two-dimensional electrophoresis, where charge separation is first used, and then these separated proteins are separated on the basis on size.

This is a very effective method for identifying a particular protein from a tissue that may contain thousands of proteins and where there may only be small differences between control and treated samples (e.g. to look for a protein involved in resistance to insect predation in plants).

Electrophoresis is a laboratory technique used to separate DNA, RNA or protein molecules based on their size and electrical charge. An electric current is used to move the molecules through a gel or other matrix. Pores in the gel or matrix work like a sieve, allowing smaller molecules to move faster than larger molecules. To determine the size of the molecules in a sample, standards of known sizes are separated on the same gel and then compared to the sample.

Electrophoresis. I worked in my dad's lab in late high school in college where we use starched electrophoresis to study population genetics. I remember first learning of and then carefully reading the paper where Linnaeus Pauling and colleagues reported using the new technology of electrophoresis in 1946, to show that sickle cell hemoglobin carries more positive charges than the normal version on its surface. Today, electrophoresis is used for so much more across biology and medicine.

Mike Smith, Ph.D.

Program Director Genome Technology Program

Division of Genome Sciences

Almost every cell in our body contains our DNA.
On average, about 99.9 per cent of the DNA between two humans is the same.
The remaining percentage is what makes us unique (unless you are an identical twin!).
Although this might sound like a small amount, it means that there are around three million base pairs that are different between two people. These differences can be compared and used to help distinguish you from someone else.
Minisatellites are short sequences (10-60 base pairs long) of repetitive DNA that show greater variation from one person to the next than other parts of the genome. This variation is exhibited in the number of repeated units or ‘stutters’ in the minisatellite sequence.
The first minisatellite was discovered in 1980.

DNA fingerprinting was invented in 1984 by Professor Sir Alec Jeffreys after he realised you could detect variations in human DNA, in the form of these minisatellites.
DNA fingerprinting is a technique that simultaneously detects lots of minisatellites in the genome to produce a pattern unique to an individual. This is a DNA fingerprint.
The probability of having two people with the same DNA fingerprint that are not identical twins is very small.
Just like your actual fingerprint, your DNA fingerprint is something you are born with, it is unique to you.

The first step of DNA fingerprinting was to extract DNA from a sample of human material, usually blood.
Molecular ‘scissors’, called restriction enzymes, were used to cut the DNA. This resulted in thousands of pieces of DNA with a variety of different lengths.
These pieces of DNA were then separated according to size by a process called gel electrophoresis:
- The DNA was loaded into wells at one end of a porous gel, which acted a bit like a sieve.
- An electric current was applied which pulled the negatively-charged DNA through the gel.
- The shorter pieces of DNA moved through the gel easiest and therefore fastest. It is more difficult for the longer pieces of DNA to move through the gel so they travelled slower.
- As a result, by the time the electric current was switched off, the DNA pieces had been separated in order of size. The smallest DNA molecules were furthest away from where the original sample was loaded on to the gel.
Once the DNA had been sorted, the pieces of DNA were transferred or ‘blotted’ out of the fragile gel on to a robust piece of nylon membrane and then ‘unzipped’ to produce single strands of DNA.
Next the nylon membrane was incubated with radioactive probes.
- Probes are small fragments of minisatellite DNA tagged with radioactive phosphorous.
- The probes only attach to the pieces of DNA that they are complementary to – in this case they attach to the minisatellites in the genome.
The minisatellites that the probes have attached to were then visualised by exposing the nylon membrane to X-ray film.
- When exposed to radioactivity a pattern of more than 30 dark bands appeared on the film where the labelled DNA was. This pattern was the DNA fingerprint.
- To compare two or more different DNA fingerprints the different DNA samples were run side-by-side on the same electrophoresis gel.

Illustration showing the steps in DNA fingerprinting.
Image credit: Genome Research Limited

DNA profiling

Modern-day DNA profiling is also called STR analysis and relies on microsatellites rather than the minisatellites used in DNA fingerprinting.
Microsatellites, or short tandem repeats (STRs), are the shorter relatives of minisatellites usually two to five base pairs long. Like minisatellites they are repeated many times throughout the human genome, for example ‘TATATATATATA’.

How is a DNA profile produced today?

DNA is extracted from a biological sample. STR analysis is incredibly sensitive so it only needs a tiny amount of someone’s DNA to produce an accurate result. As a result the DNA can be extracted from a wider range of biological samples, including blood, saliva and hair.
Unlike the original DNA fingerprinting method, DNA profiling does not use restriction enzymes to cut the DNA. Instead it uses the polymerase chain reaction (PCR) to produce many copies of specific STR sequences.
- PCR is an automated procedure that generates lots of copies of a specific sequence of DNA. It only requires small amounts of DNA to start with and can even make copies from a DNA sample that is partially degraded.
- In PCR small bits of DNA called primers bind to complementary sequences of the DNA of interest and mark the starting point for the copying of the DNA of interest.
- In STR analysis the primers used in the PCR are designed to attach to either end of the STR sequence of interest.
- The primers for each STR is labelled with a specific coloured fluorescent tag. This makes it easier to identify and record the STR sequences after PCR.
Once enough copies of the sequence have been produced by PCR, electrophoresis is used to separate the fragments according to size.
Each fragment passes by a laser which causes the fragments with fluorescent tags to glow with a specific colour. The output is displayed as a series of coloured peaks (as shown in the image below) highlighting the colour and length of each STR sequence.

Illustration showing the steps in DNA profiling.
Image credit: Genome Research Limited

The more STR sequences that are tested, the more accurate the test is at identifying someone.
Other STRs used for forensic purposes are called Y-STRs, which are derived solely from the male Y chromosome. This is useful for identifying a male perpetrator from mixed DNA samples.
Only one person in every 10 million million (10,000,000,000,000) will have a particular STR profile. With the world human population estimated at only 7,100 million (7,100,000,000) it is therefore extremely unlikely you will share the same profile as someone else, unless you are an identical twin.

Solving crime

DNA profiles are very useful in forensics because only a tiny sample of human material left behind after a crime may be sufficient to identify someone.
In the UK, a complete DNA profile consists of 11 STR sequences plus a sex determiner to confirm if the profile is from a man or a woman. Now all new profiles include an additional five STR sequences to provide consistency across borders in Europe.
In the USA, the Federal Bureau of Investigation (FBI) recommends that 13 STR sequences are tested. Many states are increasing the number of STR sequences tested to enable more efficient investigations across state borders.
A match made between a crime scene profile and an individual profile identifies a possible suspect.
A match made between different crime scene profiles indicates a repeat offender at work.
The police may use this DNA evidence to support other evidence to help prosecute someone for a crime. Complete DNA profiles give very reliable matches and may provide strong evidence that a suspect is guilty or innocent of a crime.

Illustration showing a comparison of a DNA fingerprint from a crime scene and DNA fingerprints from two suspects. The DNA fingerprint from suspect 2 matches that taken from the crime scene.
Image credit: Genome Research Limited

How are DNA profiles stored?

The UK was the first country to set up a national database of DNA profiles in 1995.
The UK National DNA Database holds the DNA profiles from a select number of UK individuals, most of which are linked to serious crimes.
The Protection of Freedom Act 2013 ensured that 1,766,000 DNA profiles taken from innocent adults and children were deleted from the UK National DNA Database.
Most countries now have a national DNA database.

Linking blood relatives

You get half of your DNA from your mother and half from your father. STRs are therefore passed down from parents to their children.
DNA profiling can be used to help confirm whether two people are related to one another and is commonly used to provide evidence that someone is, or is not, the biological parent of a child.
DNA profiling can also be used to identify victims of crime or major disasters and help bring separated families back together.
DNA profiling has a high success rate and very low false-positive rate.

Illustration comparing the DNA profiles of two parents and their child. You can see which STRs in the child have been inherited from which parent.
Image credit: Genome Research Limited