Molecular Genetic Analysis and Biotechnology - 2

Molecular Techniques Are Used for what?

Molecular Techniques Are Used to Cut and Visualize DNA Sequences

Genes need to be isolated and amplified before studied.

Specific DNA Fragments Can Be Amplified.

Ways that Specific DNA Fragments Can Be Amplified:

•Gene Cloning (in vivo): Insertion of DNA fragments into bacteria in such a way that the fragments will be stable and will be copied by the bacteria.

Polymerize Chain Reaction (in vitro): Method of enzymatically amplifying DNA fragments- most widely used molecular technique for amplifying DNA.

What does PCR do?

PCR amplifies DNA enzymatically, and DNA replication is catalyzed by a DNA polymerase.

It makes new strands of DNA, using existing strands as templates.

Taq polymerase:

DNA polymerase commonly used in PCR reactions.

•Isolated from the bacterium Thermus aquaticus, the enzyme is stable at high temperatures, so it is not denatured during the strand separation step of the cycle.

Reverse-Transcription PCR

Technique that amplifies sequences corresponding to RNA;

•Reverse transcriptase is used to convert RNA into complementary DNA, which can then be amplified by the usual polymerase chain reaction.

The Polymerase Chain Reaction (PCR) components:

•DNA template

•DNA Polymerase

•Primers

•Deoxynucleoside triphosphates (dNTPs)

•Magnesium ion (Mg2+)

•Buffer

•Water

Primers

Taq polymerase requires a primer - short sequence of nucleotides providing a starting point for DNA synthesis. 

Two primers are used - flanking the target region, binding to the template by complementary base pairing – then they’re extended by the polymerase.

Thermocycler

PCR machine = thermocycler. Regulate temperatures in a cyclical program.

The Polymerase Chain Reaction (PCR) steps:

Typical reaction - three steps.

Repeated 25-35 times, takes 2-4 hours.

Each cycle - amount of target DNA doubles.

Three Steps:

1.Denaturation (90-100°C): Heat the reaction to separate, or denature, the DNA strands. This provides the single-stranded template for the next step.

2.Annealing (30-65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.

3.Extension (72°): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.

Results of a PCR reaction are visualized using what?

Results of a PCR reaction are visualized using gel electrophoresis.

Real-Time PCR (qPCR) -

Modification of the polymerase chain reaction that is used to measure the starting amount of nucleic acid; the amount of DNA amplified is measured as the reaction proceeds.

PCR amplifies DNA fragment, and an instrument determines amount of DNA present after each cycle.

Real-time because amount of DNA amplified is measured in real-time.

Types of Real-Time PCR (qPCR) and what is it used with?

Dye-based and probe-based methods of Real-time PCR.

qPCR can be combined with RT-PCR to measure mRNA in a sample - determines level of gene expression in different cells and under different conditions = qRT-PCR or quantitative reverse transcription PCR

•Commonly used with reverse-transcription PCR (RT-PCR) to measure gene expression (qRT-PCR)..

Dye-Based RT qPCR steps

1. cDNA template is denatured

2. Dye binds to dsDNA

3. Florescence signals are detected

4. Florescence increase with more synthesized DNA

Probe-Based RT qPCR steps

1. cDNA template is denatured

2. Probe binds to target dsDNA

3. Florescence signals are detected

4. Florescence increase with more synthesized DNA

PCR

polymerase chain reaction.

•Method of enzymatically amplifying DNA fragments.

qPCR

real-time polymerase chain reaction.

Modification of the polymerase chain reaction that is used to measure the starting amount of nucleic acid; the amount of DNA amplified is measured as the reaction proceeds

RT-PCR

= reverse-transcription polymerase chain reaction.

Technique that amplifies sequences corresponding to RNA; reverse transcriptase is used to convert RNA into complementary DNA, which can then be amplified by the usual polymerase chain reaction

qRT-PCR

qRT-PCR = quantitative reverse-transcription polymerase chain reaction.

•Technique which combines RT-PCR with qPCR to enable the measurement of RNA levels through the use of cDNA in a qPCR reaction, thus allowing rapid detection of gene expression changes.

Gene Cloning:

Insertion of DNA fragments into bacteria in such a way that the fragments will be stable and will be copied by the bacteria.

Some DNA sequences are still amplified by gene cloning - alters cells so they have desired properties or produce substances of commercial value.

Need to put gene you want to clone into a cloning vector, then put gene plus vector into the bacteria, and bacteria will clone the gene.

Cloning Vectors: characteristics

An effective cloning vector has three important characteristics.

1.An origin of replication.

2.Selectable markers.

3.≥1 unique restriction sites.

Different Types of cloning vectors:

•Plasmids

•Cosmids

•Bacterial Artificial Chromosomes (BACs)

•Yeast Artificial Chromosome (YACs)

•Ti Plasmid

Plasmid Vectors:

•Plasmids: Small, usually circular DNA molecule that is distinct from the bacterial chromosome; capable of replicating independently of the bacterial chromosome. very commonly used vectors for cloning DNA fragments in bacteria

•Insert foreign DNA into plasmid using restriction enzymes.

Linkers: Small synthetic DNA fragment that contains one or more restriction sites; can be attached to the ends of any piece of DNA and used to insert it into a vector

Transformation

Transformation of host cells with plasmids.

Once DNA of interest placed inside plasmid, plasmid must be introduced into bacteria - accomplished by transformation -mechanism by which bacterial cells take up DNA from the external environment.

Screening cells for recombinant plasmids.

•Selectable markers are used to confirm which cells have been transformed.

Genes that confer resistance to an antibiotic are commonly used as selectable markers - any cell that contains such a gene will live in presence of the antibiotic, which normally kills bacterial cells.

Transformation steps

1. double stranded recombinant plasmid DNA introduced into baceterial cell

2. Cell culture produces hundreds of millions of new baceteria

3. Many copies of purified plasmid isolated from lysed bacteria

Many copies of a DNA fragment can be cloned by plasmids via Transformation.

DNA, typically in the form of a plasmid, is introduced into a competent strain of bacteria so bacteria can replicate the sequence of interest in amounts suitable for further use.

Like another type of amplification

Transformation Methods

Some types of cells undergo transformation naturally; others must be treated chemically or physically first.

Plasmids can be introduced into the cell in two ways: Chemical transformation(Incubation → heat shock →Recovery), and also electroporation (Incubation → electric shock →Recovery)

What does an idea cloning vector have?

1. An origin of replication recognized in the host cell so that it is replicated along with the DNA that it carries

2. Selectable markers - traits that enable cells containing the vector to be selected or identified

3. Third, a cloning vector needs a single cleavage site for each one or more restriction enzymes used

How are foreign DNA Fragments inserted into a plasmid?

A foreign DNA fragment can be inserted into a plasmid with the use of
restriction enzymes.

1. The plasmid and the foreign DNA are cut by the same restriction enzyme- in this case, EcoRI

2. When mixed, the sticky ends anneal, joining the foreign DNA and plasmid

3. Nicks in the sugar phosphate backbone are sealed by DNA ligase

Bacterial Transformation and Selection

Plasmids typically contains an antibiotic-resistance gene - allows bacteria to survive in the presence of a specific antibiotic.

Selection tells us we have the plasmids in the bacteria. Also need a way to select for plasmids with inserted DNA of interest. Not all plasmids will have DNA inserted.

• Place bacteria on antibiotic plate

• Grow colony

• Bacteria without plasmid die

• Each bacterium with a plasmid makes a colony

SCREENING CELLS FOR RECOMBINANT PLASMIDS

Screening cells for recombinant plasmids - identifying bacterial colonies that have successfully taken up a plasmid containing the gene of interest.

E.g. blue-white screening.

Blue colonies - plasmid is empty (no insert),  white colonies - gene of interest successfully inserted.

The lacZ gene can be used to screen for bacteria containing recombinant plasmids.

•An artificially constructed plasmid carries a fragment of the lacZ gene and an ampicillin-resistance gene.

The lacZ gene can be used to screen for bacteria containing recombinant plasmids; steps

1. Foreign DNA is inserted into the partial lacZ Gene

2. Bacteria that are lacZ- are transformed by the plasmid

3. Bacteria with an origional (nonrecombinant) plasmid produce B-galatosidase which cleaves X-gal and makes the colonies blue

4. Bacteria with a recombinant plasmid do not synthesized B-galactosidase. Their colonies remain white

5. Bacteria without a plasmid will not grow

Plasmid: Size of DNA that can be cloned, Method of Propagation, Introduction to bacteria

• As large as 15kb

• Plasmid replication

• Transformation

Phage A: Size of DNA that can be cloned, Method of Propagation, Introduction to bacteria

• As large as 23kb

• Phage reproduction

• phage infection

Cosmid: Size of DNA that can be cloned, Method of Propagation, Introduction to bacteria

• As large as 44kb

• Plasmid reproduction

• phage infection

Bacteria artificial chromosome: Size of DNA that can be cloned, Method of Propagation, Introduction to bacteria

• As large as 300kb

• Plasmid reproduction

• Electroporation

Note 1kb= 1000bp. Electroporation consists of electrical pulses that increase the permeability of a membrane

Expression Vector

Expression Vector: Cloning vector containing DNA sequences such as a promoter, a ribosome-binding site, and transcription initiation and termination sites that allow DNA fragments inserted into the vector to be transcribed and translated.

•Purpose is to introduce a specific gene into a host cell (like bacteria or yeast) and direct that cell to produce large amounts of the protein (or sometimes RNA) encoded by the gene.

Sometimes the goal in gene cloning is to produce the protein the gene encodes.

To ensure transcription and translation, a foreign gene may be inserted into an expression vector.

To ensure transcription and translation, a foreign gene may be inserted into an expression vector. Example:

1. Expression vectors contain operon sequences that allow inserted DNA to be transcribed and translated

2. They also include sequences that regulate- turn on or off-the desired gene

3. In this example, an E. coli expression vector has been used.

What else can molecular techniques be used for?

Molecular Techniques Can Be Used to Find Genes of Interest

To analyze a gene or to transfer it to another organism, the gene must be located and isolated.

DNA Libraries:

•DNA Library: Collection of clones (of genes) containing all the DNA fragments from one source.

•Genomic Library - Collection of bacterial colonies or phages containing DNA fragments that constitute the entire genome of an organism.

•cDNA (complementary DNA) Library - Collection of bacterial colonies or phages containing DNA fragments that have been produced by reverse transcription of cellular mRNA.

•Required for eukaryotic genes that will be expressed in prokaryotic cells.

DNA Sequencing

Technique for determining the sequence of bases along a DNA molecule.

DNA Sequences Can Be Determined and Analyzed

Dideoxy (Sanger) Sequencing:

•A method of sequencing DNA based on replication. The fragment to be sequenced is used as a template to make a series of new DNA molecules.

Next Generation Sequencing Technologies

•Sequencing methods that are capable of simultaneously determining the sequences of many DNA fragments; these technologies are much faster and less expensive than the Sanger dideoxy sequencing method.

•Illumina Sequencing

Third Generation Sequencing Technologies or Single-Molecule Real-Time (SMRT) Sequencing:

Determines the sequence of single molecules of DNA or RNA and allow much longer fragments to be sequenced (produce longer reads), which simplifies the assembly of fragments into a complete genome

DNA Fingerprinting (Profiling):

•Technique used to identify individuals by examining their DNA sequences.

What does the (sanger) dideoxy-sequencing reaction require?

The dideoxy-sequencing reaction requires a special substrate for DNA synthesis.

(a) Structure of deoxyribonucleoside triphosphate, the normal substrate for DNA synthesis.

(b) Structure of dideoxyribonucleoside triphosphate, which lacks an OH group on the 3′-carbon atom.

The Sanger method relies on the use of a special substrate for DNA synthesis - dideoxyribonucleoside triphosphates (ddNTPs) - terminate DNA synthesis.

What is the The dideoxy method of DNA sequencing based on?

The dideoxy method of DNA sequencing is based on the termination ofDNA synthesis.
First you amplify your DNA so you have lots of it.

Then, copies of the target DNA are isolated and split into four samples.

The dideoxy method of DNA sequencing steps

1. Each of four reactions contains: single stranded target DNA tp be sequenced, a primer, all four deoxyribonucleoside trophosphates, DNA polymerase, and one type of dideoxyribonucleoside triphosphate (ddNTP) (could be ddATP, ddCTP, ddGTP, ddTTP)

2. Nucleotides are added to the 3’ end of the primer, with the target DNA being used as a template

3. When ddNTP is incorporated into the growing chain, synthesis terminates because the ddNTP lacks a 3’-OH group

4. Synthesis terminates at different positions on different strands, which generates a set of DNA fragments of various lengths, each ending in a ddNTP with the same base

5. The fragments produced in each reaction are separated by gel electrophoresis

6. The sequence cane be read directly from the visualized bands, starting from the bottom

7. The sequence obtained us the complement of the original strand

How can the dideoxy-sequencing method be automated?

Sequencing used to be done by hand, now sequencing is usually done by automated machines that use fluorescent dyes and laser scanners.

Dideoxy-sequencing reaction is still used, but ddNTPs used in the reaction are labeled with fluorescent dyes, and a different colored dye is used for each.

dideoxy-sequencingautomated steps:

1. Single stranded DNA fragment whose base sequence is to be determined (the template) is isolated'

2. Each of the four ddNTPS is tagged with a different flourescent dye, and the dideoxy-sequencing reaction is carried out

3. The fragments that end in the same base have the same coloured dye attached'

4. The products are denatured, and the DNA fragments produced by the reaction are loaded into a single well on an electrophoresis gel. The fragments migrate through the gel according to size

5. and the fluorescent dye on the DNA is detected by a laser beam

6. Each fragment appears as a peak on the computer printout: the colour of the peak indicated which base is present

7. The sequence information is ready directly onto the computer

Illumina sequencing

Next-Generation Sequencing- Illumina sequencing is a next-generation sequencing technology.

Most next-gen sequencing technologies sequence in parallel, so hundreds of thousands or millions of DNA fragments can be sequenced at same time.

Illumina - most widely used sequencing technology.

Next-Generation Sequencing steps

1. Primers, DNA polymerase, and dNTPS are added to the flow cell. The primer attaches to the template

2. The first nucleotide is incorporated into the new strand. The tag is excited with a laser and fluoresces

3. The fluorescence is recorded by a computer. Thousands of fragments are sequences and imaged simultaneously

4. The tag and terminator are removed'

5. New dNTPs and polymerase are added

6. The next nucleotide is incorporated and excited with a laser

7. cycle repeats

DNA Fingerprinting

Technique used to identify individuals by examining their DNA sequences (DNA profiling).

STRs - People vary in the number of copies of repeats they possess at different loci.

When several different microsatellite loci are examined, the probability of two people having the same set of patterns becomes incredibly small unless they are identical twins.

Microsatellites or Short Tandem Repeats (STRs)

•Very short DNA sequence repeated in tandem.

•Detected with PCR.

•Fragments are represented as peaks on a graph:

•Homozygotes for an STR allele have a single tall peak;

•Heterozygotes have two shorter peaks.

How we can identify people based on differences in their DNA?

DNA fingerprinting can be used to identify people. Conclusion: The patterns of DNA fragments produced by individuals differ

1. DNA samples are collected and subjected to PCR

2. The length of the DNA fragment produced by PCR depends on the number of copies of the microsatellite sequence

3. The fragments are separated by gel electrophoresis. Different-sized fragments appear as different bands

What does a A DNA profile represent?

A DNA profile represents the pattern of DNA fragments produced byperforming PCR on the STR loci

Fragments are represented as peaks on a graph; the distance on the horizontal axis = size of the fragment, height of the peak = amount of DNA.

Homozygotes for an STR allele have a single tall peak; and heterozygotes have two shorter peaks.

Combined DNA Index System (CODIS).

The FBI developed a group of STR loci used for identifying people and solving crimes - Combined DNA Index System (CODIS).

Each STR locus has a large number of alleles and is located on a different chromosome = variation at each locus assorts independently.

In 2017, seven additional loci were added. 

DNA fingerprinting might be used for what purposes?

DNA fingerprinting can be used to determine the presence of a suspect ata crime scene.

DNA fingerprinting might be used to confirm that a suspect was present at the scene of a crime.

e.g Here you have two suspects profiles, and the crime scene profile.

The DNA profile of suspect 2 matches that of DNA evidence collected at the crime scene. Shown here are results from four STR loci.

What else are molecular techniques used for?

Molecular Techniques Are Increasingly Used to Analyze Gene Function

Previous techniques provided a great deal of information about the organization/nature of gene sequences, but the goal of many molecular studies is to better understand the function of these sequences

e.g Forward and Reverse Genetics

Forward Genetics

•Traditional approach to the study of gene function that begins with a mutant phenotype and proceeds to a gene that encodes the phenotype (phenotype → genotype).

e.g Mutant fly → you discover the gene responsible of red pigmentation (wild type eye colour)

Reverse Genetics

A molecular approach to the study of gene function that begins with a genotype (a DNA sequence) and proceeds to the phenotype by altering the sequence or by inhibiting its expression (genotype → phenotype).

e.g You have a wildtype (red eye) fly and you want to know what gene X does → You create a nonsense mutation and see that it causes no pigmentation in the eye

Transgenic Animals:

Another way in which gene function can be analyzed - by adding DNA sequences of interest to the genome of an organism that normally lacks them, then observe the effect of the introduced sequences on the organism’s phenotype.

Transgenic Animals:

•Transgenic: An organism permanently altered by the addition of a DNA sequence to its genome.

Transgene: Foreign gene or other DNA fragment carried by transgenic animal

Transgenic animals genomes:

Transgenic animals have genomes that have been permanently altered through recombinant DNA technology.

e.g a mouse embryo being injected with DNA.

1. Mice are mated and fertilized eggs are removed from the female mouse

2. Foreign DNA is injected into one of the pronuclei

3. Embryos are implanted in a pseudopregnant female

4. Offspring are tested for the introduced transgene

5. Mice carrying the gene are bred to produce a strain with the foreign gene

Polymerize Chain Reaction

Method of enzymatically amplifying DNA fragments.

Taq polymerase

DNA polymerase commonly used in PCR reactions. Isolated from the bacterium Thermus aquaticus, the enzyme is stable at high temperatures, so it is not denatured during the strand separation step of the cycle

Real-Time PCR (qPCR)

Modification of the polymerase chain reaction that is used to measure the starting amount of nucleic acid; the amount of DNA amplified is measured as the reaction proceeds.

Gene Cloning

Insertion of DNA fragments into bacteria in such a way that the fragments will be stable and will be copied by the bacteria.

Cloning Vector -

Stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell.

Plasmids

Small, usually circular DNA molecule that is distinct from the bacterial chromosome; capable of replicating independently of the bacterial chromosome.

Linkers

Small synthetic DNA fragment that contains one or more restriction sites; can be attached to the ends of any piece of DNA and used to insert it into a vector.

Transformation

Mechanism by which DNA found in the environment is taken up by a cell. After transformation, recombination may take place between the introduced genes and those of the bacterial chromosome.

Expression Vector -

Cloning vector containing DNA sequences such as a promoter, a ribosome-binding site, and transcription initiation and termination sites that allow DNA fragments inserted into the vector to be transcribed and translated.

DNA Library

Collection of clones containing all the DNA fragments from one source.

Genomic Library

Collection of bacterial colonies or phages containing DNA fragments that constitute the entire genome of an organism.

cDNA (complementary DNA) Library

- Collection of bacterial colonies or phages containing DNA fragments that have been produced by reverse transcription of cellular mRNA.

DNA Sequencing -

Process of determining the sequence of bases along a DNA molecule

Next Generation Sequencing Technologies -

Sequencing methods that are capable of simultaneously determining the sequences of many DNA fragments; these technologies are much faster and less expensive than the Sanger dideoxy sequencing method.

DNA Fingerprinting -

Technique used to identify individuals by examining their DNA sequences.

dideoxyribonucleoside Triphosphate (ddNTP) -

Special substrate for DNA synthesis used in the Sanger dideoxy sequencing method; identical with dNTP (the usual substrate for DNA synthesis) except that it lacks a 3′-OH group. The incorporation of a ddNTP into DNA terminates DNA synthesis.

DNA Fingerprinting -

Technique used to identify individuals by examining their DNA sequences.

Microsatellites or Short Tandem Repeats (STRs) -

Very short DNA sequence repeated in tandem.

Forward Genetics -

Traditional approach to the study of gene function that begins with a mutant phenotype and proceeds to a gene that encodes the phenotype.

Reverse Genetics

A molecular approach to the study of gene function that begins with a genotype (a DNA sequence) and proceeds to the phenotype by altering the sequence or by inhibiting its expression.