21 - Manipulating genomes Flashcards
What is the process of producing a DNA profile (genetic fingerprint) ?
1) Extract DNA
2) Digest the sample
3) Separate the DNA fragments
4) Hybridisation
5) Visualisation
What is satellite DNA?
short sequences of DNA within introns, telomeres and centromeres, that are repeated many times
What are minisatellites?
- also called variable number tandem repeats
- a sequence of 20-50 base pairs that are repeated from 50 to several hundred times
- these occur at more than 1000 locations on the human genome
What are microsatellites?
- also called short tandem repeats
- 2-4 bases repeated only 5-15 times
What is the location of satellites? How do they vary between individuals?
-Satellites always appear in the same locations on a chromosome
-but the number of repeats varies between individuals
-The more closely 2 people are related, the more similar their satellites should be
Why are introns not used usually?
Coding DNA (exons) is not used because in reality, in most people the coding genome is very similar, so a unique pattern would not be gained.
What is DNA profiling used for?
DNA profiles can be used to identify individuals, establish genetic relationships and compare relatedness
What is DNA profiling?
Producing an image of the patterns in DNA
What is the first step of DNA Profiling?
Extraction of DNA
- initially large samples of DNA was needed, but now only the tiniest of fragments is needed and PCR is used to make large amounts of the sample
What are the steps of PCR? Describe them.
1) Denaturation (96 °C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
2)Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
3)Extension (72 °C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
This cycle repeats 25 - 35 times in a typical PCR reaction
What is Taq polymerase?
PCR requires a DNA polymerase enzyme that makes new strands of DNA. The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus).
Why is Taq polymerase used?
T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-stable and is most active around 70°C (a temperature at which a human or E. coli DNA polymerase would be non-functional). This heat-stability makes Taq polymerase ideal for PCR. As we’ll see, high temperature is used repeatedly in PCR to denature the template DNA, or separate its strands.
What is a PCR primer?
a short sequence of nucleotides that provides a starting point for DNA synthesis.
PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length. Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied. The primers bind to the template by complementary base pairing.
When the primers are bound to the template, they can be extended by the polymerase,
What is the second step of DNA profiling?
Digest the sample: Cut it into smaller sections using enzymes known as restriction endonucleases.
How is the sample in DNA profiling digested?
-Restriction endonucleases are used to cut the DNA into smaller pieces
-They cut the DNA at locations known as restriction/recognition sites which have specific nucleotide sequences (allowing the enzyme to recognise the site)
-The enzymes make 2 cuts, one on each strand, breaking the molecule apart
-A mixture of different restriction endonucleases are used which each target specific restriction sites
-They will choose a combination of enzymes that leave satellite DNA intact, therefore the resulting fragments of DNA contain a mixture of intact satellite sections.
Why do different restriction endonucleases cut at different sites?
Because the shape of a specific recognition sequence is complementary to the active site of that enzyme. This enables many different satellites to be cut out.
What is the third step of DNA profiling
To produce a pattern that can be analysed, the fragments must be separated from one another
This is done through gel electrophoresis: a technique that utilises that uses the way charged particles move through a gel medium
Explain gel electrophoresis
A sheet of agarose gel is produced (roughly 0.5-1cm thick) using agarose powder and a buffer solution (which maintains pH)
The gel will have a row of small wells at one end
The gel is placed into a tank containing more buffer
The digested DNA samples containing satellites are deposited into the wells
A “Ladder” is added to one of the empty wells. This contains DNA fragments of known lengths, and is used as a reference to compare the other fragments to.
On one end of the tank (near the wells) is the negative cathode, at the other end is the positive anode
DNA is negatively charged due to the phosphate groups and so will move through the gel towards the anode when an electric current is flowing
Agarose gel has a 3D mesh-like structure
DNA has to pass through this mesh to get towards the anode
Small DNA fragments pass though the gel faster than larger fragments
This means that when the current is turned off at the end of the process, the smaller fragments will be closer to the anode than the larger fragments
What is the fourth step of DNA profiling?
Hybridisation: Radioactive or fluorescent probes are added to the DNA fragments to bind with them.
What is southern blotting?
- the agarose gel is immersed in alkali in order to separate the DNA double strands into single strands (ssDNA).
The single stranded DNA fragments are transferred to nitrocellulose paper of nylon membrane and is covered by absorbent paper.
This draws the alkaline solution containing the DNA through the membrane by capillary action.
The ssDNA is pulled up along with the solution and is caught by the paper/membrane as it can’t pass through it
UV light or 80C heat is the applied, which fixes the DNA in place on the sheet in the same positions they were in the gel
What are DNA probes?
These are radioactive or fluorescent short DNA or RNA sequences that are complimentary to known DNA sequences in the microsatellites
They bind to complimentary strands under certain conditions of pH or temperature
- excess probes have been washed off
What is the 5th step of DNA profiling?
Visualisation of evidence: X-ray images are taken to create a DNA profile.
Describe the process of gene ‘Sanger’ sequencing
Sanger sequencing uses radioactive (1) modified bases, terminators / ddNTPs (1) to terminate elongation of the DNA strand (1). Use four different reaction mixtures, each one containing a different ddNTP. (1) The primers anneal to the template strand and DNA polymerase can then bind to the dsDNA (1). As the strand is copied by DNA polymerase, the ddNTPs are incorporated randomly (1) so that many different fragment sizes are produced, each terminating with a certain ddNTP (1). Run the four different samples on an electrophoresis gel to separate the different fragments on the basis of size (1). Visualise using radioactive film (1). Read the bands – each band will show a particular fragment ending in a particular ddNTP
What is Sanger sequencing, and what is its primary purpose in molecular biology?
Sanger sequencing is a DNA sequencing method used to determine the nucleotide sequence of a DNA molecule. It is essential for identifying genetic sequences and understanding genetic variation.
How does Sanger sequencing work at the molecular level?
Sanger sequencing relies on the incorporation of chain-terminating dideoxynucleotides (ddNTPs) into a growing DNA strand, resulting in fragments of varying lengths that can be separated by size and analyzed.
What is the significance of the four chain-terminating dideoxynucleotides (ddNTPs) used in Sanger sequencing?
The four ddNTPs (ddATP, ddTTP, ddCTP, and ddGTP) are used to halt DNA strand synthesis at specific points, enabling the determination of the DNA sequence.
How are DNA fragments separated and visualized in Sanger sequencing?
DNA fragments are separated by size using gel electrophoresis, and the separated fragments are visualized by exposing them to a fluorescent dye and detecting the emitted fluorescence.
In Sanger sequencing, why is a primer required to initiate DNA synthesis?
A primer is needed to initiate DNA synthesis because DNA polymerase requires a starting point (primer) with a free 3’-OH group to add nucleotides.
What is the role of DNA polymerase in Sanger sequencing?
DNA polymerase is used to replicate the DNA template by incorporating regular deoxynucleotides (dNTPs) and chain-terminating ddNTPs into the growing DNA strand.
How is the DNA sequence determined during Sanger sequencing?
The DNA sequence is determined by analyzing the order of ddNTPs that terminate the DNA fragments in each of the four separate reactions, corresponding to each base (A, T, C, and G)
What are some applications of Sanger sequencing in biology and research?
Sanger sequencing is used for DNA sequencing of specific genes, identifying mutations, studying genetic disorders, and validating PCR products, among other applications.
What are some limitations of Sanger sequencing compared to more recent sequencing methods?
Sanger sequencing is relatively slow, labor-intensive, and less cost-effective for large-scale sequencing projects compared to Next-Generation Sequencing (NGS) methods.
How do terminator bases work in Sanger sequencing?
These are modified nucleotides that lack the 3’ hydroxyl (-OH) group, which is necessary for DNA strand elongation. In Sanger sequencing, each of the four terminator bases (ddATP, ddTTP, ddCTP, and ddGTP) is used in separate reactions, alongside their regular deoxynucleotide counterparts.
when a DNA polymerase enzyme encounters a terminator base (ddNTP), it cannot add more nucleotides to the growing DNA strand because the 3’ hydroxyl group required for the formation of phosphodiester bonds is absent. As a result, the DNA synthesis process is prematurely terminated at the site where the terminator base is incorporated.
What does DNA synthesis in sanger sequencing produce?
This process generates a set of DNA fragments of different lengths, with each fragment ending at the position where a terminator base was incorporated.
What is capillary sequencing, and how does it relate to DNA sequencing?
Capillary sequencing is a modern DNA sequencing technique that uses capillary electrophoresis to separate DNA fragments. It is a high-throughput method commonly used for DNA sequencing
How is DNA sequencing accomplished in capillary sequencing, and what is the role of capillaries in the process?
In capillary sequencing, DNA fragments are separated in thin capillaries filled with a gel matrix. As the fragments move through the capillary, they are separated by size, enabling the determination of the DNA sequence.
What advantages does capillary sequencing offer over traditional Sanger sequencing?
Capillary sequencing is faster, more automated, and provides higher throughput compared to traditional Sanger sequencing. It is well-suited for large-scale DNA sequencing projects.
How are DNA fragments labeled for detection in capillary sequencing?
Fluorescent dyes are commonly used to label DNA fragments. Each of the four nucleotide bases is typically labeled with a different fluorescent dye, allowing for their identification during electrophoresis.
How is the DNA sequence determined in capillary sequencing, and what data is generated?
The DNA sequence is determined by analyzing the order of the fluorescently labeled peaks corresponding to the four nucleotide bases as they pass through the capillary. This data generates a sequencing trace.
Explain capillary sequencing
In capillary Sanger Sequencing, four different terminators are used (A, C, T and G). Each is labelled with a different fluorescent dye. Due to this, only 1 tube needs to be used and all four terminators can be used at once.
The DNA is put at the end of a long thin capillary tube filled with agarose gel
A current is passed though the gel causing the DNA fragments move towards the negative electrode
The smaller fragments move more quickly than the larger ones so the fragments separate in the capillary tube (longest at the beginning, shortest at the end
Because each terminator base has its own fluorescent colour, lasers can be used to distinguish which bases are present, and in which order
The sequence can then be read using lasers and light sensors
How is the genome of an organism sequenced?
Only small sections of a genome can be sequenced at once
1,000s of BPs at a time
The Human genome is 3 Billion BPs long
To produce a full genome sequence these sections need to be patched together to complete the genome
This is done by identifying overlapping sectio
What is a ddNTP?
A dideoxynucleotide triphosphate. A dNTP (free nucleotide with 3 phosphates) missing an oxygen on its 3rd carbon. This makes it unable to form phosphodiester bonds. When it is added by DNA polymerase this terminates DNA synthesis. They are also known as terminator bases and are usually tagged with a radioactive or fluorescent dye.
What is Next-generation sequencing?
Any method of DNA sequencing that has replaced the Sanger method is referred to as nextgeneration sequencing (NGS)
Why is NGS more desirable than older methods?
NGS methods can be one thousand times faster than older methods of sequencing
* The reduction in time required for sequencing means that costs are also greatly reduced
o NGS methods cost roughly 0.1% of the cost of chain-termination methods
What is nanopore technology?
determines the order of nucleotides in DNA or in RNA by measuring fluctuations in an electric current as the molecule passes through a nanopore.
How does nanopore technology work?
- The nanopore is embedded in a membrane that separates two chambers containing electrolyte solutions
- voltage is applied, an enzyme steadily ratchets the molecule through the nanopore along
-its sequence based is on how much short sequences of individual nucleotides block the flow of ions and tiny changes in electrical current.
What is the human genome project?
- an international research program involving over 1000 scientists. It was a publicly
funded project, aiming to map and understand all the genes in the human genome
-The Human Genome Project determined there are just over 20,000 human protein-coding genes. reflects how complex the regulation of these genes has to be
What was the main purpose of the human genome project?
The main purpose of the human genome project was to identify the entire genetic makeup of humans. It would help
in identifying genes involved in various genetic diseases.
