DNA Technologies

Question 1

DNA technologies

Answer 1

• DNA sequencing and profiling can be used to determine the sequence of genes along a strand of DNA

DNA Sequencing

• Determines the precise order of nucleotides in a sample of DNA
• Techniques include Sanger, Massively Parallel Sequencing, and nanopore sequencing

DNA profiling

• A distinct series of bands, representing an organism’s unique DNA profile, is created. This is often done using gel electrophoresis

Question 2

PCR (Polymerase chain reaction) overview

Answer 2

• Used to amplify DNA: make many copies of the piece of DNA being studied
• A sample containing the DNA of interest is mixed with oligonucleotide primers (one forward and one reverse), Taq DNA polymerase, and free nucleotides. The mixture is then placed in a thermal cycler, which subjects it to multiple cycles of alternating temperature to stimulate amplification
• DNA replication occurs in each cycle, so after every cycle, the amount of DNA in the sample is doubled. After more than 40 cycles, one target molecule of DNA will have produced more than a million molecules

Benefits

• Amplification creates large volumes of DNA from a small initial amount of DNA. This allows thorough research and testing to be carried out on the sample, as high volumes of DNA are typically needed
• PCR is used in conjunction with sequencing and profiling in genetic testing for diseases like Huntington, and paternity tests

Question 3

Substances used in PCR

Answer 3

Template DNA

• The original strand of DNA is needed to begin the process of amplification, as all copies of the DNA will be made from it

Taq DNA Polymerase

• Like regular DNA polymerase, it synthesises nucleotides on the template strand

Oligonucleotide primers

• These are short, single-stranded DNA fragments. They bind to DNA sequences on either side of the region of DNA to be copied
• They signal Taq where to begin DNA synthesis
• One forward and one reverse primer are needed

Free nucleotides

• Needed so that Taq can synthesise a new complementary strand during DNA synthesis.

Question 4

Thermal cycling (PCR)

Answer 4

1. Denaturing

• The reaction mixture is heated to 96C. Hydrogen bonds are broken by the high temperature, causing the double-stranded DNA to denature into two single strands ⭐

2. Annealing

• The mixture is cooled to 50C, allowing oligonucleotide primers to pair with the DNA template
• The cooler temperature is essential as it enables hydrogen bonds to form between complementary bases, and the higher quantity of primers mean the strands bind to primers instead of the opposite strand.

3. Extension

• Mixture is warmed to 60C, so Taq can synthesise nucleotides
• Taq recognises an olignucleotide primer as a starting point for synthesis, and fills in nucleotides in the correct places along the DNA template to form a new complementary strand

⭐ This step is basically carrying out the function of helicase,

Question 5

DNA sequencing

Answer 5

Process of determining the precise order of nucleotides in a sample of DNA

• The mixture containing sample DNA also contains artificial nucleotides (terminating nucleotides) that are tagged with coloured fluorescent dyes. Each type of terminating nucleotide (A, T, C, G) are tagged with a different colour
• During PCR DNA replication, Taq polymerase may by chance use a terminating nucleotide. This would stop DNA synthesis on that strand.
• Termination of synthesis is random, so many different lengths of DNA fragments can be produced
• After PCR is done, the DNA fragments are then placed in an apparatus that performs capillary electrophoresis. A laser beam is shone at the fragments, causing the terminating nucleotides to fluoresce.
• The colour and intensity of the fluorescence is analysed by a computer to determine the sequence of DNA bases.

Question 6

DNA Profiling

Answer 6

Process of determining an individual’s DNA characteristics

• 99.9% of DNA is shared among humans, but variable regions in the non-coding sections of DNA can vary widely between individuals, and hence be used to identify people
• Genetic markers, often located within these variable regions, can be used to study evolutionary relationships or identify individuals/species
• Genetic markers are polymorphic within the population: they show variation. Examples are SNPs and STRs (short tandem repeats)

Question 7

STRs (Short Tandem Repeats)

Answer 7

• Short sections of DNA with repeating nucleotides
• Each person has 2 copies of every STR, inherited maternally and paternally. If the length of an STR is the same on each chromosome, the individual is homozygous for that STR.

Steps of DNA profiling

1. DNA is extracted from the sample
2. DNA is added to PCR for amplification
3. Electrophoresis gel used to detect in size differences of the STRs by registering peaks on the computer

E.g. in one person, AT can be repeated 5 times. In another person, it can be repeated 7 times at that exact location on a chromosome

Question 8

Electrophoresis producing fragments

Answer 8

• DNA can be cut into fragments using restriction enzymes, which cleave DNA at specific recognition sites
• Restriction enzymes cut DNA at different sites in different individuals due to the different sequence of bases each person possesses
• Restriction techniques are used in tandem with PCR to amplify the target alleles

Question 9

Electrophoresis gel

Answer 9

Separates DNA fragments based on their length

1. Agarose gel is immersed in a buffer solution to stabilise pH and promote electrical conductivity
2. DNA is mixed with a fluorescent dye which attaches to the fragments of DNA
3. DNA is placed into wells in the gel
4. Voltage is applied, causing the DNA fragments to migrate through the gel towards the positive terminal
5. Longer fragments are heavier and hence move slower than lighter, shorter fragments. This causes DNA to sort itself into groups based on length, as the shortest will have travelled the furthest
6. After a time, remove the gell from solution and place it on a UV light box. The length of DNA fragments can be estimated by comparing them to the standard DNA ladder

The different DNA sequences between individuals results in a unique banding shown on a gel for every individual. This banding pattern is the DNA profile

Question 10

Dideoxynucleotides

Answer 10

Nucleotides that are engineered to give off a “colour” signal that can be read by a computer. This is used to determine the sequence of a DNA segment.

• Dideoxynucleotides have been modified to lack a hydroxyl group at the 3’ carbon, so that they terminate DNA synthesis when they are incorporated into a strand of DNA
• Used in the Sanger method

Question 11

Use of gel electrophoresis for disease diagnosis

Answer 11

Disease and disorders can be identified through gel electrophoresis. This method of DNA profiling can be used to determine a specific gene, not just STR markers.
E.g. Huntington’s disease is marked by more repeats in the HD gene. Using a DNA ladder, the bp of the gene can be identified, and if this mutation has occurred.

Question 12

Different uses of DNA Profiling

Answer 12

1. Using 13 STR (known to be different) to compare genomes of individuals
2. To compare a specific part of DNA i.e. hereditary diseases, other disorders.