PCR Electrophoresisby Mike Belmonte, Muskegon Michigan

Edited by C. Kohn, Waterford, WI

Objectives Describe the technique that enables scientists to mass-produce specific segments of DNA in a test tube. Describe a technique used to compare DNA samples.

Key Terms polymerase chain reaction (PCR) gel electrophoresis genetic marker DNA fingerprint

DNA ProfilingHow to solve a crime or determine a parent based on DNA

We’re very similar!

99.9% of human DNA is exactly the same from person to person◦As a species, we are more like each other than most

other living species. Sidebar: racism is not at all scientific – one race cannot be

biologically better than another if we are 99.9% the sameHowever, the 0.1% difference between

individuals equates to 3 million bases that are different between you and the person next to you. ◦This is not enough to make them very different from

you.◦This is enough to distinguish your DNA from their’s

DNA Profiling

DNA Profiling is when a geneticist examines the 3 million base pairs that could be different for any variations that are unique to an individual. ◦This is also know as DNA Fingerprinting

When we determine how your DNA is different from everyone else’s, we create a genetic profile that is specific only to you.

Steps of DNA Profiling

To create a DNA fingerprint, we have four steps:◦1. collect a sample of DNA◦2. amplify specific regions of the DNA that

typically have a lot of variety from person to person

◦3. count repeated sequences (STRs)◦4. look for matches from collected samples

(e.g. blood drops)

Step 1: Collect a sample

Let’s imagine we have a crime scene and we want to match blood found on the scene to a suspect.

If the suspect drank from a cup, the saliva on the cup would have a tiny amount of cheek cells. ◦This is enough to create a profile.

We would collect DNA from the crime scene as well as DNA from the cells in the saliva on the cup.

Step 2: Amplify DNA Regions

Next, we use Polymerase Chain Reaction (PCR) to amplify specific regions (we’ll cover this process later in this PPT). ◦PCR can make billions of identical copies from a

single snippet of DNAGeneticists look for Short Tandem

Repeats◦STRs are short units of DNA (4-5 bases) that

repeat over and over in a stretch of DNA.

STRs

STRs are critical to gene research because they are the source of most of the variation in DNA

Individual A might have 20 more STRs for one region than Individual B. This creates a basis for comparing which DNA samples came from which people (or suspects)

In this case, STR1 has 7 repeats

STR2 has 4 repeats at the same spot.

3. The PCR process (gene copying) allows us to see to compare how long each STR is. ◦ In criminal forensics, we look at 13 STR regions

4. We then look for a match. ◦ If all 13 STR regions match, we know that the DNA found

at the scene of a crime is from that person.◦ The odds of it being from someone else are 1 in 1 billion.

STR Region# Repeats

(Crime Scene Sample) # Repeats (Suspect 1) # Repeats (Suspect 2) 1 7 8 72 4 11 43 14 13 144 11 18 115 8 7 8… … … …

Polymerase Chain ReactionA copy machine for DNA

Mass-Producing DNA in a Test Tube

The polymerase chain reaction (PCR) is a technique that makes many copies of a certain segment of DNA without using living cells.

Starting with a single DNA molecule, PCR can generate 100 billion identical copies of a gene in just a few hours.

A great advantage of PCR is that it can copy one specific segment of DNA from within a tremendous amount of DNA.

The technique is so precise and powerful that its starting material does not even have to be purified DNA.

PCR Animations 1, 2, 3, 4, 5•PCR works by separating the dual-strand of DNA into two individual strands so that each side can then be primed (by primers) and copied by polymerase enzymes.

•This process is repeated over and over.

•It can amplify a specific sequence of DNA by as many as one billion times!

Taq Polymerase

For PCR, we use a special polymerase enzyme called the Taq Polymerase

The Taq polymerase was discovered in bacteria in a hot spring such as those you’d find in Yellowstone Park)

The Taq polymerase can work in very hot conditions that would denature normal proteins (including enzymes like Polymerase)

Taq Polymerase

A heat-resistant polymerase enzyme is necessary because of the constant re-heating of the DNA to denature it.

The process involves constant heating, cooling, and re-heating.

This would destabilize any normal polymerase enzyme.

Taq does everything a normal polymerase would do (i.e. add complementary bases), but remains stable at very high temperatures.

Only a tiny amount (less than one millionth of a gram) of DNA needs to be present in the starting material.

Using PCR to produce multiple copies of a DNA sample can make analysis of that sample much easier. ◦ It’s easier to investigate something if

there is a lot of it!

It also enables scientists to make copies of very rare DNA. ◦ For example, scientists have used the

technique to clone DNA pieces recovered from… 5,000-year-old human remains A 40,000-year-old woolly mammoth

frozen in a glacier A 30-million-year-old plant fossil.

PCR also has applications in medicine. ◦ For example, PCR makes it possible to

detect viral genes in cells infected with the virus that causes AIDS.

PCR Steps

•Often scientists want to compare PCR results of different DNA samples.

•One useful technique for sorting molecules or fragments of molecules by length is called gel electrophoresis.

Comparing DNA

Gel Electrophoresis

First, each DNA sample is cut up into fragments by a group of restriction enzymes (chemical scissors)

Remember that restriction enzymes recognize and cut within specific DNA sequences.

These specific DNA sequences are located at different places along the strand of DNA, depending on the source.

Because of this, each DNA sample will have a unique banding pattern.

DNASeq. 1

DNASeq. 2

The same restriction enzyme will cut the same gene from different people in different ways.

Restriction Fragments

For example, EcoRI is a restriction fragment that always looks for the GAATTC sequence.

It will always cut DNA between the G and the first A.

This sequence is likely to show up multiple times in a normal DNA segment.

Because this segment will occur different places in different DNA, it will create uniquely sized ‘chunks’ of DNA for each person.

Gel Electrophoresis

Next, a few drops of each DNA sample are placed in a small pocket, or well, at one end of a thin slab of gelatin-like material called a gel. The other end of the gel has a positive charge.

All DNA molecules are negatively charged (DNA has a negative charge because of the phosphate ions in its chemical "backbone“) , so they move through pores in the gel toward the positive pole.

Gel ElectrophoresisThe shorter DNA

fragments slip more easily through the pores of the gel.

After a set period of time, the shorter DNA fragments will travel farther through the gel.

The shorter fragments will be closer to the positive end of the gel than the longer fragments.

In the last step, the gel is treated with a stain that makes the DNA visible under ultraviolet light. The DNA fragments show up as a series of bands in each "lane" of the gel. Each band consists of many DNA fragments of one particular size.

The pattern of bands will be different for each of the four samples because each sample has a unique set of DNA fragment lengths.

Gel Electrophoresis Animations 1, 2 ,3, 4, 5, 6

DNA Fingerprinting 1, 2, 3 Just as the skin patterns on

your fingertips make up your particular set of fingerprints, you have a particular banding pattern produced by your restriction fragments called a DNA fingerprint. ◦ The fingerprint is created by a

combination of your STRs at different regions and where the restriction enzyme cut the DNA.

◦ STR creates the size of the band◦ Restriction Enzyme creates the

number of the bands

Unless you have an identical twin, no one else is likely to have the exact same DNA fingerprint as you.

Genetic markers can help to pick out the differences between 2 DNA fingerprints.

A genetic marker is a gene that is found at a known location and produces a predictable pattern.

Genetic markers can occur in alleles for diseases or other traits. ◦ Markers can also be located in one of the many noncoding

stretches of the human genome (introns). ◦ Introns account for about 97% of the human

genome.

When DNA fingerprints are used in court as evidence, genetic markers from these highly variable noncoding regions are used.

Why is this important? ◦ To use DNA to identify a specific person

accurately, you want to compare genetic markers that are unlikely to be shared with any other person. Introns are least likely to be repeated among

different people

Using PCR and gel electrophoresis, a DNA fingerprint can be made from cells in a single drop of blood or from a hair follicle. DNA is extracted from the small sample, and multiple copies are made using PCR.

Genetic markers (usually from introns) are then compared.

In most cases, the probability of two people having identical genetic markers is small—somewhere between one chance in 100,000 and one chance in 1,000,000,000 (one billion), depending on the number of genetic markers compared.

Concept Check 13.4

1. Name one application of the mass-production of DNA using PCR.

2. Explain how gel electrophoresis compares DNA samples.

3. Why are genetic markers from noncoding regions useful in distinguishing DNA fingerprints?

Extra Resources

Gel Electrophoresis Virtual LabDNA Detective; Who Dun It ?Polymerase Chain Reaction Quiz 1, 2, 3DNA Fingerprinting Tutorial & Quiz (advan

ced)DNA Fingerprint Quiz 1, 2 ,3 , 4, 5