Chapter 4: DNA Structure and Analysis

What is the relationship between genes, proteins, and function?

Molecular biology bridges the disciplines of genetics, which is the study of genes, and biochemistry, which includes the study of protein function. Essentially, genes are translated into proteins , and these proteins then produce the traits or phenotypes that ultimately represent the organism's function

What is the central dogma – the flow of genetic material?

genetic information contained in DNA is transmitted to RNA, and then the information in RNA is transferred to protein⁵. This relationship is summarized by the phrase: DNA to RNA to protein to trait.

Exception to Centeal Dogme

Reverse Transcription (RNA → DNA):

Process: RNA acts as a template to create DNA, using the enzyme reverse transcriptase from retroviruses

What occurs during transcription?

Transcription is the process where the information contained in DNA is transferred into an RNA molecule. During this step, an enzyme called RNA polymerase copies one of the DNA strands to create a molecule of messenger RNA (mRNA).

What occurs during translation?

Translation is the process where the information contained in the RNA molecule is transferred into a protein. In this process, the sequence of bases on the mRNA is read, and that information is converted into a polypeptide chain composed of amino acids, which subsequently folds into a protein

What is the role of DNA polymerase?

The enzyme DNA polymerase is responsible for copying both DNA strands and making a brand new copy of the DNA in a process known as DNA replication

What is the role of RNA polymerase?

RNA polymerase is the enzyme that copies one DNA strand into messenger RNA (mRNA) during the process of transcription

What happens when RNA to DNA (remember viruses)?

For exmaple retroviruses like (HIV), genetic information can flow from RNA back into DNA. This process is carried out by an enzyme called reverse transcriptase, which has become an important tool in cloning.

What are the three parts of a nucleotide?

DNA strands are polymers of nucleotides, and each individual nucleotide consists of three main parts:

• sugar (deoxyribose),

• a phosphate group, and

• one of four nitrogenous bases, which are adenine (A), thymine (T),guanine (G), and cytosine (C).

Does DNA have a positive or negative charge?

DNA fragments are negatively charged, which is why they are repelled from the negative electrode (cathode) and move toward the positive electrode (anode) when placed in an electric field, such as during gel electrophoresis.

ompare and contrast DNA and RNA (shape, nitrogen bases, sugars, locations).

DNA is typically a double-stranded molecule where the two strands are oriented in opposite directions/ contains the nitrogenous base thymine (T)/ DNA carries the genetic information that is inherited

RNA: which is a single-stranded molecule/ (mRNA) has the same sequence as the DNA sense strand but uses the base uracil (U) instead of thymine/while RNA transfers that information for protein production.

If the DNA sequence is ATCGATCC, then what is the complementary DNA sequence?

In DNA, adenine (A) always pairs with thymine (T), and cytosine (C) always pairs with guanine (G). Therefore, the complementary DNA sequence for ATCGATCC would be UACGUACG,

If the DNA sequence is ATCGATCC, then what is the complementary RNA sequence?

When DNA is transcribed, the resulting messenger RNA (mRNA) sequence is complementary to the template strand but substitutes uracil (U) for thymine (T). Assuming the provided sequence is the sense strand, the complementary RNA sequence will be UACGUACG,

dentify the leading and lagging strands during replication

one new strand (leading strand) to be built continuously towards the replication fork, while the other (lagging strand) is built discontinuously in short Okazaki fragments away from the fork, both in the 5' to 3' direction, requiring RNA primers and DNA ligase for joining. 

Describe how DNA is built (direction and what part of the nucleotide the new nucleotide is added to)

Nucleic acids are synthesized in vivo (in living organisms) exclusively in a 5 to 3 direction. As the strand grows, each new nucleotide is added to the 3v hydroxyl group of the sugar, which is the group attached to carbon 3 of the existing nucleotide.

What is genetic engineering? What is recombinant DNA?

Genetic engineering is the process where scientists cut out a DNA fragment from one organism and insert it into a different organism.

Recombinant DNA is the resulting molecule (thing thst occurs) that has been assembled by linking DNA fragments from different organisms

What are restriction enzymes?

Restriction enzymes, are bacterial enzymes that cut DNA in very predictable locations. They are foundational tools in molecular biology because they recognize a specific sequence of base pairs, called a restriction site, and chemically separate the DNA molecule at that location.

How are restriction enzymes used in biotechnology?

Restriction enzymes are used to enable scientists to move genes from the genome of one organism into the genome of another, a necessary step for genetic engineering. They cut the DNA by hydrolyzing the sugar-phosphate bond between two specific nucleotides in the restriction site on each strand

What is the function of ligase?

Ligases are enzymes that join, otwo pieces of DNA back together by reforming the phosphate bonds that were broken by restriction enzymes.

In the lab, if restriction enzymes have created DNA fragments with complementary sticky ends, ligase fuses the backbone while hydrogen bonds hold the fragments in position.

What does CRISPR stand for

CRISPR is an acronym that stands for Clustered Regularly Interspaced Short Palindromic Repeats

How does the CRISPR-Cas9 system work?

like a precise molecular "find and cut" tool for DNA, using a guide RNA (gRNA) to direct the Cas9 enzyme (molecular scissors) to a specific DNA sequence, where Cas9 makes a double-stranded cut, allowing scientists to disable, correct, or insert genes by leveraging the cell's own repair mechanisms

How is CRISPR used in agriculture, industry, health, and medicine? In agriculture,

RISPR accelerates the development of improved crops and livestock with desirable traits such as faster growth, higher nutrient content, and disease resistance.

• Industry, a modified CRISPR-Cas9 system has been used to create a yeast strain to produce lipids and polymers that could be useful in the development of biofuels, adhesives, and fragrances.

• public health, the technology is being experimented with to engineer "gene drives" to spread specific genes through insect pest populations, such as mosquitoes carrying malaria or the Zika virus, to cause them to die or become infertile.

• medicine, researchers are looking to CRISPR as a technique for editing out genetic defects that cause diseases like sickle cell disease and cystic fibrosis, as well as for developing more targeted and effective cancer treatments.

Be able to show how to make TAE buffer, Fast Blast, and agarose gels.

The most common electrophoresis buffers for DNA, such as Tris/acetic acid/EDTA (TAE) and Tris/boric acid/EDTA (TBE), are supplied as concentrated stock solutions and must be diluted to a $1\times$ working solution before use^45454545. To prepare an agarose gel, agarose powder is mixed with the $1\times$ electrophoresis buffer, the mixture is heated to melt the agarose, and then the molten agarose is cooled to approximately $55^{\circ}C$⁴⁶. The cooled, molten agarose is poured into a gel tray that has a comb inserted to form the wells for loading the DNA samples

ow does gel electrophoresis work? Be sure to know the equipment involved.

Agarose gel electrophoresis separates DNA fragments by size using an electric field.

The equipment includes the agarose gel (which acts as a molecular sieve), an electrophoresis chamber filled with a conductive buffer solution (like TAE or TBE), and a power supply that passes a direct current between wire electrodes at each end of the chamber.

Since DNA fragments are negatively charged, they are repelled from the negative electrode (cathode) and move toward the positive electrode (anode).

o smaller or larger DNA fragments travel farther

Smaller DNA fragments travel farther and faster than larger ones because the rate at which a DNA fragment migrates through the gel is inversely proportional to its size in base pairs

What stain is used to visualize the results?

he agarose gel must be stained with an appropriate dye to make the separated DNA bands visible. Safer positive stains commonly used include Fast Blast or fluorescent dyes like SYBR® Safe DNA stain

If you are using 10× loading dye and adding it to a 50 $\mu$L reaction for a total of 60 $\mu$L, how much 10× loading dye do you need to add to make the final dilution 1×?

To make the final dilution $1\times$ in a total volume of $60~\mu L$ using a $10\times$ stock, you need $6~\mu L$ of the $10\times$ loading dye. This is calculated by finding the volume of stock that is $\frac{1}{10}$ of the final volume: $V = (1\times \times 60~\mu L) / 10\times = 6~\mu L$.

hat percent agarose gels are typically used? Describe how you would make one

separating relatively large DNA fragments (greater than 1 kilobase or kb), the typical percentage of agarose used is $0.7\%$ to $1\%$ (mass/volume)⁵³.

To create the gel, you would mix the appropriate amount of agarose powder with a $1\times$ electrophoresis buffer, heat the mixture until the agarose melts, and then cool the molten agarose to approximately $55^{\circ}C$⁵⁴. The cooled, molten agarose is then poured into a gel tray that has a comb inserted to form the wells^55555555.

hat safety issues should be addressed when conducting gel electrophoresis?

electric shock from the direct current, requiring the power source to be unplugged and turned off before the lid of the chamber is removed or the gel is handled.

staining dyes used to visualize the DNA, as some common stains are mutagens and require UV-blocking eyewear and UV light shielding.

What does DNA fingerprinting mean?

DNA fingerprinting is a forensic technique that uses DNA analysis to establish a pattern unique to an individual. This unique pattern can then be used in criminal investigations to either include or exclude a suspect.

What are RFLPs and how are they used? RFLP stands for Restriction Fragment Length Polymorphisms.

These are variations in the size of DNA fragments among individuals that result from differences in the location of restriction sites. RFLPs are used to create a unique banding pattern for an individual's DNA, serving as the basis for the technique known as DNA fingerprinting.

What is the purpose of each lab?

• 4.A Restriction Site Prediction is designed to predict restriction sites using the bioinformatics tool NEBcutter.

• 4.B Casting Agarose Gels is for practicing the preparation of agarose gels.

• 4.C Dye Electrophoresis allows for the visualization of how electrophoresis separates materials.

• 4.D Restriction Digestion and Analysis of Lambda DNA involves practicing restriction enzyme digestion and analyzing the results using agarose gel electrophoresis.

• 4.E Forensic DNA Fingerprinting provides practice with the techniques used for forensic analysis.

• 4.F Plasmid Mapping is used to determine the arrangement of restriction sites on a circular piece of DNA called a plasmid.