Tema 5

• The majority of DNA sequences in genomes have unknown functions, including some that code for proteins.
• Sophisticated techniques exist to locate, purify, and manipulate genes from large chromosomes to study the function of small DNA sequences.
• This helps to understand gene function in normal conditions and the molecular bases of disease.

Techniques

• Techniques are used for isolating, amplifying, and manipulating small DNA segments for further study.

DNA Cloning

• DNA cloning typically involves separating a small DNA segment (like a specific gene) and joining it to a small carrier DNA molecule called a bacterial plasmid.
• This produces a recombinant DNA molecule capable of replicating many times within a cell, generating identical copies of the fragment.
• This process achieves selective amplification of a gene or essential segment within a living host cell.

Vectors

• Vectors are used in DNA cloning as carriers for DNA segments.

Bacterial Plasmids

• Bacterial plasmids are small, circular, double-stranded DNA molecules capable of providing antibiotic resistance to the host cell.
• They possess properties that make them suitable as cloning vectors, including:
  • Existing as single or multiple copies and replicating independently of the bacterial DNA, like episomes. Episomes are extra-chromosomal replicating genetic elements that can replicate autonomously.
  • They can also be inserted into the host organism's chromosome and replicate with it, using the host's replication machinery.
  • Having a known complete DNA sequence and a precise location of restriction enzyme cutting sites (multi-cloning site), allowing for insertion and extraction of the DNA to be cloned.
  • Being easily isolatable from the host cell chromosome due to their small size.

Plasmid structure

• ORI: Origin of replication
• Plasmid cloning vector
• Multi Cloning Site: A region with multiple sequences that can be cut with specific enzymes.
• Basic plasmids cannot function as expression plasmids.

Steps in DNA Cloning

• DNA cloning involves specific steps to isolate and amplify a desired DNA fragment.

Cutting and Pasting DNA

• This requires restriction endonucleases (restriction enzymes) and DNA ligases.
• Restriction endonucleases recognize and cut at specific sequences (restriction sites) or a predictable pattern nearby.
• They are found in bacteria, which use them as defense against foreign viral DNA.
• Both the fragment of interest and the bacterial plasmid (or chosen vector) are cut.
• This cut can produce:
  • Cohesive ends: These are short, single-stranded fragments that protrude and facilitate joining to other complementary fragments cut by the same enzyme.
  • Blunt ends: These ends lack unpaired bases and are more difficult to ligate.
• DNA ligase enzymes covalently join the newly synthesized fragments during replication, catalyzing the formation of a phosphodiester bond.
• In vitro, DNA ligases can be used with recombinant DNA technology to join cut ends produced by restriction enzymes.

Transformation

• Introduction of plasmids or other types of DNA into bacteria.
• To facilitate incorporation of recombinant DNA, bacteria are subjected to stress such as high temperatures.

Selection (Insertion)

• Selecting bacteria that contain recombinant DNA.
• Plasmids contain a gene for antibiotic resistance, allowing bacteria to survive in the presence of a specific antibiotic like ampicillin.
• The bacteria are cultured in a medium containing the specific antibiotic.
• Bacteria without the plasmid die.
• Bacteria with the plasmid will form colonies, meaning groups of bacteria that are clones of the first, thus containing the plasmid for resistance, and therefore, the DNA of interest.
• Colonies serve as factories for plasmid cloning.

Culturing Selected Bacteria

• Selected bacteria are cultivated in large quantities to be used as "factories" for plasmid cloning or protein coding.
• The plasmids and encoded proteins are then harvested and purified.

Types of Plasmids

• Plasmids are designed for either cloning or expression.

Cloning Plasmids

• Cloning plasmids do not necessarily need to be expressed.

Expression Plasmids

• Expression plasmids are used in industry and are cloning vectors equipped with transcription and translation signals for expressing the gene of interest.
• They contain necessary sequences like promoters.
• To avoid introns, the gene that is transcribed with introns and exons is taken and matured into a mature messenger inside the cell. In the lab, this is done by isolating RNA and describing it with retrica.
• Inside the cell, the cDNA is cloned in expression vectors.
• In the laboratory, this is achieved through reverse transcription:
  • Isolation of mRNA.
  • Addition of reverse transcriptases that digest it, while one strand of DNA is synthesized.
  • Synthesis of the DNA chain complementary to the first.
  • Obtaining cDNA (genes of DNA without introns, similar to mRNA).

Applications of DNA Technology

• DNA technology has various significant applications.

Gene Function Study

• Used to study gene functions by varying sequences through mutation to observe their function or involvement.

Gene Therapy Tools

• Produces therapy vectors containing a copy of the non-defective gene for a disease.
• The gene is expressed in the diseases cells.

Protein Production

• Used to generate proteins in abundance for therapy, such as insulin for diabetics, using E. coli.

Generating DNA or cDNA Libraries

• A gene library is a collection of DNA fragments, genome (DNA), or transcriptome (cDNA) that has been cloned into vectors.
• Libraries provide a large number of small fragments spanning the entire genome for sequencing.
• They are collections that contain the entire genome or transcriptome of an organism and were important in early sequencing efforts.
• DNA libraries are the same in all cells of an organism, whilst RNA libraries vary depending on the cell type.

Creating DNA Libraries

DNA Libraries

• Partial digestion of the total DNA with a restriction enzyme that cuts frequently, but it cuts partially leaving larger fragments, to preserve intact genes.
• Cloning in large cloning vectors.
• Bacterial transformation, obtaining clones that contain all the sequences of the genome.

cDNA Libraries

• mRNA cannot be cloned directly; therefore, cDNA must be made as a double strand.
• No digestion is required because the DNA fragments are small and can be directly cloned into cloning/expression vectors.
• Transformation of bacteria, obtaining clones containing the DNA of the starting cells (different cDNA in different cell types).

PCR (Polymerase Chain Reaction)

• It is a technique for replicating DNA, producing millions of copies in a test tube, without cells (in vitro).
• Generates enough DNA to be used for sequencing, digestion with restriction enzymes, or cloning in a plasmid.
• PCR amplifies a specific DNA region of interest.
• Specificity comes from using two oligonucleotide primers that hybridize to complementary sequences on opposite DNA strands, flanking the sequence of interest.

PCR Steps

• PCR consists of repeated cycles of denaturation, primer annealing, and extension with DNA polymerase, exponentially amplifying segments.

Denaturation (96°C)

• The DNA sample is heated to separate the two DNA strands.

Primer Annealing (55-65 °C)

• The reaction is cooled to allow excess primers to hybridize to the DNA strands.

Extension (72 °C)

• Each strand is copied by a DNA polymerase, starting at the primer sites in the presence of the four dNTPs.
• Heat-stable polymerases like Taq polymerase (from thermophilic bacteria) are used, as they do not denature at the high temperatures required for DNA denaturation.
• Only DNA sequences from 50 bp to 10 kb can be amplified, because polymerase cannot finish the reaction.

Visualization of results

• Agarose gel electrophoresis is used to visualize PCR products.
• It involves applying an electric current to a gel matrix containing pores, through which DNA moves at a rate inversely proportional to its size.
• The DNA fragments are separated by size, allowing classification based on the number of base pairs.
• A marker of known molecular weight determines the size of the fragments in the PCR sample.
• Millions of DNA molecules of the same size will form a band in the gel.
• The gel is stained with a dye that binds to DNA to reveal the bands.

DNA Sequencing

• Involves determining the nucleotide sequence (A, T, C, and G) of a DNA fragment.
• There are two main types of DNA sequencing methods.

Sanger Sequencing

• Developed by Frederick Sanger in 1977, capable of sequencing DNA regions up to 900 base pairs long.
• Although slow, it was used for the Human Genome Project in 2001.
• Used to sequence short DNA fragments (cloning or PCR fragments) of up to 900 bp.

Sanger Sequencing Ingredients

• DNA template to be sequenced.
• DNA polymerase.
• A primer (single-stranded DNA) that acts as an initiator of the polymerase.
• Four deoxynucleotides (dNTPs: dATP, dTTP, dCTP, dGTP).
• Dideoxynucleotides (ddNTPs): chain terminators which, because they have an H in place of OH 3', prevent the reaction from continuing.
• The four ddNTPs (ddATP, ddTTP, ddCTP, ddGTP) are labelled with different fluorochromes and are needed in smaller quantities than dNTPs.

Sanger Sequencing process

• Desnaturalization of two DNA strands
• Primer annealing to only one primer
• Extension: The process continues until the polymerase encounters a ddNTP, preventing the reaction from continuing.
• Sequences of different lengths are obtained following ddNTP incorporation at different positions (multiple cycles until all have been occupied).
• Since the nucleotides are labelled, one can know their occupied positions. These will be those that are sequenced.

Sequencing Output: Chromatogram

• A chromatogram is a graphical (or other type of) representation of a detector's response.
• The peaks in a chromatogram indicate the fluorescence intensity of each base; the distinct peaks correspond to nucleotides.
• If there are two peaks for one base, this could indicate a heterozygote.
• The Human Genome Project used Sanger sequencing to determine the sequences of relatively small human DNA fragments.
• The fragments were aligned based on overlapping regions to assemble larger DNA regions and, ultimately, complete chromosomes.
• The whole process required 13 years and 3 billion dollars.
• The last portion of sequencing is to organize the sequences.
• With sequences that overlap, an informatic program lines up sequences that match.

Next-Generation Sequencing

• The new methodologies on a larger scale will increase the speed and reduce costs of DNA sequencing.
• Used to sequence genomes and exomes.
• Allow massive parallel sequencing, meaning sequences can start from one sample and millions of DNA molecules can be sequenced at the same time.
• Does not need electrophoresis in agarose gel.
• Able to identify which nucleotides are being incorporated at each moment.
• Sequences genomes at a reasonable price and in a quick manner.