DNA Technology and Genomics

Biologists have developed techniques for artificial manipulation of DNA, cells, and organisms.

Challenges of Studying DNA

The size of DNA molecules poses a significant challenge.
Naturally occurring DNA is very long, and specific genes may comprise a small portion (e.g., $1/100,000$ ) of the chromosome.
There may only be a small difference in the surrounding nucleotides.

Restriction Enzymes

Restriction enzymes are molecular scissors.
They recognize DNA sequences 4-6 bp in length and cut DNA molecules at specific locations.
In nature, these enzymes help protect bacteria and archaea from foreign DNA viral attacks.
There are many different types of restriction enzymes.
Each restriction enzyme is very specific and recognizes a short DNA sequence known as a restriction site.
The DNA is cut at specific sites within the DNA strand.
Restriction enzymes cut DNA in repeatable ways.
Restriction enzymes that produce sticky ends are desirable.
A mathematical problem associated with the use of restriction enzymes: the larger the genome, the more cut sites there will be.

PCR (Polymerase Chain Reaction)

PCR is used to prepare a large quantity of DNA when only a small amount is present.
It can quickly generate a large amount of DNA from a small amount.
A 3-step cycle brings about a chain reaction that produces exponential growth of identical DNA molecules.
- A. The solution containing the piece of DNA is heated to break the H-bonds and separate the DNA into single strands.
- B. DNA primers added to the mixture anneal to the DNA strands.
- C. The heat-stable Taq polymerase adds nucleotides to the primers in the standard 5’à3’ direction synthesizing the target sequence.
Specificity is a key benefit. If a target segment is identified and a primer is made to it, then only a small amount is necessary to start.
The primer will only replicate the target segment because this is all they are able to bind to.
After just a few cycles, a very large amount of the target segment will be identified.

Tandem Repeat DNA

Short non-coding DNA sequences that repeat in a tandem pattern of one or more nucleotides is called a tandem repeat.
The STRs can be cut with restriction enzymes.
Tandem repeats are useful in paternity testing, forensic science, and other forms of DNA profiling.
Polymorphisms in chromosomal DNA can arise from the presence of these repeats.
The numbers of repeats varies from person to person and is unique for each individual.
It serves as a molecular marker that can be used to provide a lot of useful information.
Using PCR to amplify the DNA at particular sites yields fragments of DNA of varying lengths depending on how many repeated segments are contained within the fragment.
Variation in the number of these tandem repeats leads to differences between individuals.

Restriction Fragment Analysis

Treating the DNA with restriction enzymes and then running the samples through a gel enable researchers to produce banding patterns characteristic of the starting molecule and the restriction enzyme(s) used to treat the DNA.
This analysis can be used to examine bacterial chromosomes for certain genes, settle paternity suits, and to provide evidence to crime scene investigators.
Analysis of these repeats provides a powerful tool for scientists to analyze differences in the nucleotide sequences of DNA molecules.

Restriction Fragment Length Polymorphisms (RFLPs)

When non-coding regions of DNA were treated with restriction enzymes and banded, scientists discovered differences in non-coding regions on homologous chromosomes.
These serve as genetic markers of non-coding DNA that appears near a particular locus in a genome.
There are many RFLP variants within a population.
RFLP data is often used in crime investigations because the likelihood that two individuals will have the same banding patterns are vanishingly minuscule at best.
These are variations within the restriction sites of individuals within a population.
Variation between 2 individuals occurs in about 1 out of every 1000bp.
Our genome is about $3.2 \text{ billion}$ bp.
That means 2 individuals will vary by more than $3 \text{ million}$ bp.
These changes disrupt the restriction site and create the unique genetic fingerprint.

Key Differences Between RLFP and STR Analysis

RFLPs look at differences in nucleotide sequences, while STRs look at repeat segments within the DNA.
RFLP analysis relies on restriction enzymes.
STR analysis relies on PCR to amplify and identify the variations within the number of repeat sequences.

Gel Electrophoresis

Agarose gel separates DNA fragments by size and charge.
The fibers in the gel separate the fragments; smaller fragments migrate farther than the larger fragments.
The negatively charged DNA fragments migrate toward the positive pole of the electrophoresis box.
Treating the DNA with various restriction enzymes and manipulating it in a variety of ways can give a lot of information.

Southern Blot Analysis

Scientists use a Southern Blot to Analyze RFLPS or Regular DNA.
Gel electrophoresis and the analysis of restriction fragments is used in crime scene investigations and paternity suits all the time.
Scientists can use restriction fragment information to determine if a person has a particular disease.
Scientist can also use RFLP information to determine the likelihood of inheriting a certain genetic disease.

DNA Sequencing

Sequencing became the biggest drawback when the HGP was moving forward. There wasn’t a fast way to sequence the genes. The initial challenge was to develop new technologies to quickly sequence the DNA.
Frederick Sanger developed the first method to sequence the genes called the dideoxy chain-termination method.

The Dideoxy Chain-Termination Method

A set of complementary DNA strands are synthesized from an original DNA strand.
Each strand starts with the same primer and ends with a modified nucleotide that is fluorescently labeled and lacks a hydroxyl group; it is called a dideoxyribonucleotide.
The dideoxyribonucleotide terminates the growing DNA strand because it lacks a 3’- OH group.
In the newly synthesized strands, each nucleotide position along the original sequence is represented by strands ending at that point with the complementary ddNTP.
Each type of ddNTP, tagged with a distinct fluorescent label, identifies the ending nucleotides of the new strands, ultimately revealing the sequence of the DNA.

3 Main Steps of the Dideoxy Method

1. Millions of fragments of DNA to be sequenced are denatured into single strands and incubated in a test tube with a primer that will base pair with the known 3’ end of the template stand, DNA polymerase, A,T,C,&G and the 4 ddNTP’s which are tagged with a specific fluorescent molecule.
2. Synthesis of the new strand starts at the 3’ end of the primer and continues until a ddNTP is added at random. This prevents further elongation. Eventually, a labeled set of strands of various lengths is generated
3. The labeled strands in the mixture are separated by passage through a polyacrylamide gel in a capillary tube; the shorter the strands move through faster than the larger ones.
- A fluorescent detector can sense the color of each tag as the strands come through. Strands that differ in as little as 1 nucleotide can be distinguished.
The color of the fluorescent tag on each strand indicates the identity of each nucleotide at its end, and the results can be printed out on a spectrogram and the sequence, which is complementary to the template strand, can then be read from the bottom to top.

Uses of Whole Genome Sequencing

Whole genome sequencing has a wide variety of uses. The increasing speed of this process and the reduced cost are leading to more uses.
- Diagnosis of disease
- Understanding the genetic basis of some diseases (cancer)
- Epidemiology
- Studying evolution

Transposable Elements

Transposable elements are known as “jumping genes.”
They have the ability cut themselves out of a genome and move themselves to another portion of the genome.

DNA as Hereditary Material

So how do we know that DNA is the hereditary material?
From the standpoint of the nature of science, the answer to this question is quite interesting.

Determining the Chemical Composition of DNA

After Morgan determined that genes were on chromosomes, scientists began wondering what they were made of.
Early on, scientists thought they were made of proteins because they were large and structurally sound.
Nucleic acids were deemed too simple to carry out many of the complex tasks chromosomes required.
As scientists continued their experiments with viruses and bacteria, many results were observed that gave support to the notion that DNA was the hereditary material.
A classic experiment demonstrated the genetic role of DNA.

Frederick Griffith's Experiment

Studied the bacterium that caused pneumonia—S. pneumonia.
Worked with two strains: pathogenic (S) and non-pathogenic (R).
(S) smooth cells produce mucous capsules that protect the bacteria from an organism’s immune system—pathogenic.
(R) rough cells have no mucous capsule and are attacked by an organism’s immune system—non-pathogenic.
Griffith mixed heat-killed pathogenic (S) bacteria with living non-pathogenic (R) bacteria, the non-pathogenic (R) bacteria began producing the mucous capsule and became pathogenic (S).
The new bacteria that arose from the bacteria were somehow transformed into pathogenic S. pneumonia.
Griffith called this process transformation.
This experiment did not identify DNA as the transforming factor, but it set the stage for other experiments.

Avery, McCarty, and McCleod

They worked for about 10 years trying to identify the transforming factor.
After isolating and purifying numerous macromolecules from the heat killed pathogenic bacteria, they could only get DNA to work.
The prevailing beliefs about proteins vs. DNA continued to generate skepticism.

The Hershey-Chase Experiment

In 1952, Alfred Hershey and Martha Chase performed experiments with viruses showing that DNA is genetic material.
Viruses (aka phages) are DNA or RNA wrapped in a protein.
E. coli is a bacteria that is often used in experiments.
Hershey and Chase used the T2 phage because it was generally accepted to be DNA wrapped in protein.
E. coli was used because it was easily obtainable and was readily attacked by T2.
They sought to determine whether or it was DNA or protein that was the hereditary material.
Their experiment demonstrated which part of the T2 entered the E. coli.
They grew T2 in the presence of radioactive sulfur—proteins contain sulfur, DNA does not.
Next, they grew the T2 in a separate batch of radioactive phosphorous. The DNA of T2 contains phosphorous—the proteins do not.
The scientists now had 2 batches of T2, one labeled with radioactive sulfur, and one labeled with radioactive phosphorous.
These 2 batches were separately incubated with non-radioactive samples of E. coli and analyzed shortly after infection.
Shortly after infection, the E. coli samples were spun in a blender to knock off loose parts of T2.
The mixtures were then spun in high-speed centrifuges for a long time to separate out various parts of the mixture.
At the bottom of the tube was a pellet of E. coli.
The pellet was examined for radioactivity and radioactive phosphorous was found.
The supernatant was analyzed and a lot of radioactive sulfur was found, but no radioactive phosphorous.
This indicates that the DNA got into the E. coli and was in the pellet
The protein did not get into the bacteria and was left in the supernatant.
Furthermore, when the bacteria in the pellet were plated on culture medium, they produced more T2 containing radioactive phosphorous.
They concluded:
- That the virus injects DNA into the E. coli and it is the genetic material that programs the cells to produce new T2 phages.
- The protein stays outside.
This experiment provided firm evidence that DNA was the hereditary material and not protein.

Mutations

Mutations result from a change in the DNA sequence.
They can be caused by errors in DNA replication and/or repair.
They can also be caused by environmental factors such as chemicals, radiation or pathogens.
Mutagens cause mutations.
Mutations are completely random, but some base pairs are more prone to mutating than others.
Cytosine, for example is prone to mutation when it becomes methylcytosine.
5-methylcytosine is the methylated form of cytosine. This can affect expression patterns of associated genes.
5-methylcytosine can also lose its amino group and become thymine. This is another form of the cytosine mutation.
Some mutations are caused by spontaneous chemical reactions.
For instance, removing the amino group and forming a keto group below converts C to U.
Some mutations occur when chemicals bind to the DNA.
For instance, benzopyrene binds to G and when the DNA polymerase reaches the modified G, it can’t read it and randomly inserts one of the four bases resulting in a mutation.
Mutations occur in the genome and can be silent, can disrupt the function of proteins, and can create new variations of alleles.
Substitutions, insertions, and deletions are common genetic mutations.

Substitutions

Substitutions may or may not result in a change in the protein.
The redundancy of the genetic code may result in a silent mutation.
This particular mutation changes the amino acid and ultimately the protein.
This is an example of an SNP.
This substitution mutation results in a missense mutation.
This example results in a nonsense mutation and an immediate stop.
Substitution mutations can produce single nucleotide polymorphisms (SNPs).
SNPs can also refer to changes in the genetic sequence of non-coding DNA. It is often used in crime scene analysis and other forms of forensic analysis.

Insertions and Deletions

Insertions and deletions disrupt the reading frame resulting in a missense or nonsense mutation. Some involve a single nucleotide.
Insertions and deletions can also involve any number of base pairs.
Insertions and deletions shift the reading frame resulting in a missense or nonsense mutation.

Chromosomal Mutations

These mutations affect large sections of a chromosome. Includes:
- Inversion
- Deletion
- Duplication
- Insertion
- Translocation

Germline vs. Somatic Mutations

Germline mutations can result in the formation of new alleles, or birth defects. Germline mutations can be inherited.
Somatic mutations can result in cancer.

Gene Knockout

Gene knockout is a technique used to determine the function of a particular gene.
Knowing the steps is not important, but understanding how the information can be used is.
There is a library of knockout organisms that can be used as models in research.

CRISPR

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats.
It has a wide variety of uses and the potential for misuse.
Palindromic segments are part of the CRISPR sequence.
The PAM sequence is required for the Cas9 to cut the DNA within the protospacer sequence. It will make the cut 3 base pairs away from the PAM.
CrRNA + tracRNA (trans-activating CRISPR RNA) create a scaffold region and a 20-nucleotide guiding region.
A Single Guide RNA (sgRNA) also contains a 20-nucleotide guiding region and a scaffold region.
Cas9 differentiates between bacterial DNA (does NOT cut) and viral DNA (cut).
Ethical issues should be considered regarding CRISPR.