Conclusion - DNA and Biotechnology - MCAT Biochemistry Review

MCAT Biochemistry Review

Chapter 6: DNA and Biotechnology


In this chapter, the DNA molecule was discussed. The importance of this molecule as an archive in the cell was highlighted. The unique structure of the DNA double helix and complementary base-pairing are integral to DNA molecules' ability to replicate and pass information from cell to cell and from generation to generation. Replication of DNA is a complex process involving many enzymes and proteins that are highly coordinated to ensure efficiency. The discoveries of DNA structure and function aid us in understanding how cellular processes take place but also provide us with many tools to exploit in the laboratory.

DNA is one of the most heavily tested concepts on the MCAT. This makes sense because not only has our understanding of the molecule increased exponentially over the last few decades, but also our ability to manipulate DNA. This has led to the creation of an entire industry of biotechnology that will assuredly grow during your career as a medical student and physician. In the next chapter, we turn to DNA's counterpart, RNA. We'll explore how the genes discussed in this chapter can actually turn into functional proteins through the key processes of transcription and translation.

Concept Summary

DNA Structure

· Deoxyribonucleic acid (DNA) is a macromolecule that stores genetic information in all living organisms.

· Nucleosides contain a five-carbon sugar bound to a nitrogenous base; nucleotides are nucleosides with one to three phosphate groups added.

o Nucleotides in DNA contain deoxyribose; in RNA, they contain ribose.

o Nucleotides are abbreviated by letter: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).

· DNA is organized according to the Watson–Crick model.

o The backbone is composed of alternating sugar and phosphate groups, and is always read 5′ to 3′.

o There are two strands with antiparallel polarity, wound into a double helix.

o Purines (A and G) always pair with pyrimidines (C, U, and T). In DNA, A pairs with T (via two hydrogen bonds) and C pairs with G (via three hydrogen bonds). RNA does not contain thymine, but contains uracil instead; thus, in RNA, A pairs with U (via two hydrogen bonds).

o Purines and pyrimidines are biological aromatic heterocycles. Aromatic compounds are cyclic, planar, and conjugated, and contain 4n + 2 π electrons (where n is any integer; Hückel's rule). Heterocycles are ring structures that contain at least two different elements in the ring.

o Chargaff's rules state that purines and pyrimidines are equal in number in a DNA molecule, and that because of base-pairing, the amount of adenine equals the amount of thymine, and the amount of cytosine equals the amount of guanine.

o Most DNA is B-DNA, forming a right-handed helix. Low concentrations of Z-DNA, with a zigzag shape, may be seen with high GC-content or high salt concentration.

· DNA strands can be pulled apart (denatured) and brought back together (reannealed). Heat, alkaline pH, and chemicals like formaldehyde and urea can cause denaturation of DNA; removal of these conditions may result in reannealing of the strands.

Eukaryotic Chromosome Organization

· DNA is organized into 46 chromosomes in human cells.

· In eukaryotes, DNA is wound around histone proteins (H2A, H2B, H3, and H4) to form nucleosomes, which may be stabilized by another histone protein (H1). As a whole, DNA and its associated histones make up chromatin in the nucleus.

o Heterochromatin is dense, transcriptionally silent DNA that appears dark under light microscopy.

o Euchromatin is less dense, transcriptionally active DNA that appears light under light microscopy.

· Telomeres are the ends of chromosomes. They contain high GC-content to prevent unraveling of the DNA. During replication, telomeres are slightly shortened, although this can be (partially) reversed by the enzyme telomerase.

· Centromeres are located in the middle of chromosomes and hold sister chromatids together until they are separated during anaphase in mitosis. They also contain a high GC-content to maintain a strong bond between chromatids.

DNA Replication

· The replisome (replication complex) is a set of specialized proteins that assist the DNA polymerases.

· To replicate DNA, it is first unwound at an origin of replication by helicases. This produces two replication forks on either side of the origin.

o Prokaryotes have a circular chromosome that contains only one origin of replication.

o Eukaryotes have linear chromosomes that contain many origins of replication.

· Unwound strands are kept from reannealing or being degraded by single-stranded DNA-binding proteins.

· Supercoiling causes torsional strain on the DNA molecule, which can be released by DNA topoisomerase II (DNA gyrase), which creates nicks in the DNA molecule.

· DNA replication is semiconservative: one old parent strand and one new daughter strand is incorporated into each of the two new DNA molecules.

· DNA cannot be synthesized without an adjacent nucleotide to hook onto, so a small RNA primer is put down by primase.

· DNA polymerase III (prokaryotes) or DNA polymerase α and δ (eukaryotes) can then synthesize a new strand of DNA; they read the template DNA 3′ to 5′ and synthesize the new strand 5′ to 3′.

o The leading strand requires only one primer and can then be synthesized continuously in its entirety.

o The lagging strand requires many primers and is synthesized in discrete sections called Okazaki fragments.

· RNA primers can later be removed by DNA polymerase I (prokaryotes) or RNase H (eukaryotes), and filled in with DNA by DNA polymerase I (prokaryotes) or DNA polymerase δ (eukaryotes). DNA ligase can then fuse the DNA strands together to create one complete molecule.

DNA Repair

· Oncogenes develop from mutations of proto-oncogenes, and promote cell cycling. They may lead to cancer, which is defined by unchecked cell proliferation with the ability to spread by local invasion or metastasize (migrate to distant sites via the bloodstream or lymphatic system).

· Tumor suppressor genes code for proteins that reduce cell cycling or promote DNA repair; mutations of tumor suppressor genes can also lead to cancer.

· During replication, DNA polymerase proofreads its work and excises incorrectly matched bases. The daughter strand is identified by its lack of methylation and corrected accordingly.

· Mismatch repair also occurs during the G2 phase of the cell cycle, using the genes MSH2 and MLH1.

· Nucleotide excision repair fixes helix-deforming lesions of DNA (such as thymine dimers) via a cut-and-patch process that requires an excision endonuclease.

· Base excision repair fixes nondeforming lesions of the DNA helix (such as cytosine deamination) by removing the base, leaving an apurinic/apyrimidinic (AP) site. An AP endonuclease then removes the damaged sequence, which can be filled in with the correct bases.

Recombinant DNA and Biotechnology

· Recombinant DNA is DNA composed of nucleotides from two different sources.

· DNA cloning introduces a fragment of DNA into a vector plasmid. A restriction enzyme (restriction endonuclease) cuts both the plasmid and the fragment, which are left with sticky ends. Once the fragment binds to the plasmid, it can be introduced into a bacterial cell and permitted to replicate, generating many copies of the fragment of interest.

o Vectors contain an origin of replication, the fragment of interest, and at least one gene for antibiotic resistance (to permit for selection of that colony after replication).

o Once replicated, the bacterial cells can be used to create a protein of interest, or can be lysed to allow for isolation of the fragment of interest from the vector.

· DNA libraries are large collections of known DNA sequences.

o Genomic libraries contain large fragments of DNA, including both coding and noncoding regions of the genome. They cannot be used to make recombinant proteins or for gene therapy.

o cDNA libraries (expression libraries) contain smaller fragments of DNA, and only include the exons of genes expressed by the sample tissue. They can be used to make recombinant proteins or for gene therapy.

· Hybridization is the joining of complementary base pair sequences.

o Polymerase chain reaction (PCR) is an automated process by which millions of copies of a DNA sequence can be created from a very small sample by hybridization.

o DNA molecules can be separated by size using agarose gel electrophoresis.

o Southern blotting can be used to detect the presence and quantity of various DNA strands in a sample. After electrophoresis, the sample is transferred to a membrane that can be probed with single-stranded DNA molecules to look for a sequence of interest.

· DNA sequencing uses dideoxyribonucleotides, which terminate the DNA chain because they lack a 3′ –OH group. The resulting fragments can be separated by gel electrophoresis, and the sequence can be read directly from the gel.

· Gene therapy is a method of curing genetic deficiencies by introducing a functional gene with a viral vector.

· Transgenic mice are created by integrating a gene of interest into the germ line or embryonic stem cells of a developing mouse.

o Organisms that contain cells from two different lineages (such as mice formed by integration of transgenic embryonic stem cells into a normal mouse blastocyst) are called chimeras.

o Transgenic mice can be mated to select for the transgene.

· Knockout mice are created by deleting a gene of interest.

· Biotechnology brings up a number of safety and ethical issues, including pathogen resistance and the ethics of choosing individuals for specific traits.

Answers to Concept Checks

· 6.1

1. Nucleosides contain a five-carbon sugar (pentose) and nitrogenous base. Nucleotides are composed of a nucleoside plus one to three phosphate groups.

2. A pairs with T (in DNA) or U (in RNA), using two hydrogen bonds. C pairs with G, using three hydrogen bonds.

3. DNA contains deoxyribose, while RNA contains ribose. DNA contains thymine, while RNA contains uracil. Usually, DNA is double-stranded, while RNA is single-stranded.

4. The aromaticity of nucleic acids makes these compounds very stable and unreactive. Stability is important for storing genetic information and avoiding spontaneous mutations.

5. This does not violate Chargaff's rules. RNA is single-stranded, and thus the complementarity seen in DNA does not hold true. For single-stranded RNA, %C does not necessarily equal %G; %A does not necessarily equal %U.

· 6.2

1. The five histone proteins are H1, H2A, H2B, H3, and H4. H1 is the only one not in the histone core.





Density of chromatin packing


Not dense (uncondensed)

Appearance under light microscopy



Transcriptional activity



3. High GC-content increases hydrogen bonding, making the association between DNA strands very strong at telomeres and centromeres.

· 6.3







Unwinds DNA double helix

Single-stranded DNA-binding protein


Prevents reannealing of DNA double helix during replication



Places∼10-nucleotide RNA primer to begin DNA replication

DNA polymerase III


Adds nucleotides to growing daughter strand

DNA polymerase α


Adds nucleotides to growing daughter strand

DNA polymerase I


Fills in gaps left behind after RNA primer excision

RNase H


Fills in gaps left behind after RNA primer excision

DNA ligase


Joins DNA strands (especially between Okazaki fragments)

DNA topoisomerase II (DNA gyrase)


Reduces torsional strain from positive supercoils by introducing nicks in DNA strand

2. The lagging strand is more prone to mutations because it must constantly start and stop the process of DNA replication. Additionally, it contains many more RNA primers, all of which must be removed and filled in with DNA.

3. Telomeres are the ends of eukaryotic chromosomes and contain repetitive sequences of noncoding DNA. These protect the chromosome from losing important genes from the incomplete replication of the 5′ end of the DNA strand.

· 6.4

1. Oncogenes (or, more properly, proto-oncogenes) code for cell cycle-promoting proteins; when mutated, a proto-oncogene becomes an oncogene, promoting rapid cell cycling. Tumor suppressor genes code for repair or cell cycle-inhibiting proteins; when mutated, the cell cycle is allowed to proceed unchecked. Oncogenes are like stepping on the gas pedal, mutated tumor suppressor genes are like losing the brakes.

2. The parent strand is more heavily methylated, whereas the daughter stand is barely methylated at all. This allows DNA polymerase to distinguish between the two strands during proofreading.


Repair Mechanism

Phase of Cell Cycle

Key Enzymes/Genes

DNA polymerase (proofreading)


DNA polymerase

Mismatch repair


MSH2, MLH1 (MutS and MutL in prokaryotes)

Nucleotide excision repair

G1, G2

Excision endonuclease

Base excision repair

G1, G2

Glycosylase, AP endonuclease

4. Nucleotide excision repair corrects lesions that are large enough to distort the double helix; base excision repair corrects lesions that are small enough not to distort the double helix.

· 6.5

1. Genomic libraries include all of the DNA in an organism's genome, including noncoding regions. This may be useful for studying DNA in introns, centromeres, or telomeres. cDNA libraries only include expressed genes from a given tissue, but can be used to express recombinant proteins or to perform gene therapy.

2. PCR increases the number of copies of a given DNA sequence and can be used for a sample containing very few copies of the DNA sequence. Southern blotting is useful when searching for a particular DNA sequence because it separates DNA fragments by length and then probes for a sequence of interest.

3. Dideoxyribonucleotides lack the 3′ –OH group that is required for DNA strand elongation. Thus, once a dideoxyribonucleotide is added to a growing DNA molecule, no more nucleotides can be added because dideoxyribonucleotides have no 3′ –OH group with which to form a bond.

4. Transgenic mice have a gene introduced into their germ line or embryonic stem cells to look at the effects of that gene; they are therefore best suited for studying the effects of dominant alleles. Knockout mice are those in which a gene of interest has been removed, rather than added.

Shared Concepts

· Biochemistry Chapter 4

o Carbohydrate Structure and Function

· Biochemistry Chapter 7

o RNA and the Genetic Code

· Biology Chapter 1

o The Cell

· Biology Chapter 2

o Reproduction

· Biology Chapter 3

o Embryogenesis and Development

· Biology Chapter 12

o Genetics and Evolution