L8: Molecular Biology Methods:DNA

Question 1

What is nucleic acid hybridisation, and why is it important?

Answer 1

Nucleic acid hybridisation is the process where nucleic acids with complementary sequences anneal or bind together.

Question 2

Why is nucleic acid hybridisation important

Answer 2

This principle is fundamental to various molecular biology techniques like PCR, Sanger sequencing, next-generation sequencing, and array CGH, as it allows for the specific detection and analysis of DNA or RNA sequences.

Question 3

How do probes and primers function in nucleic acid hybridization?

Answer 3

Probes/primers are short, single-stranded DNA molecules designed with specific sequences that hybridize to their complementary sequences in a target nucleic acid. Probes are used to detect specific DNA sequences, while primers are used in PCR to initiate the synthesis of new DNA strands.

Question 4

What is Polymerase Chain Reaction (PCR)?

Answer 4

PCR is a method used to amplify a specific region of DNA, generating large amounts of DNA from a limited number of copies.

Question 5

Why is PCR important

Answer 5

It is crucial for DNA fingerprinting, genetic diagnostics, and early cancer detection due to the ability to amplify large amounts of a specific DNA region

Question 6

What is PCR based on

Answer 6

The knowledge of DNA replication in vivo

Question 7

What are the key components required for PCR?

Answer 7

• Thermostable DNA polymerase (e.g., Taq DNA Polymerase)
• DNA template
• dNTPs + Mg²⁺
• Two oligonucleotide primers

Question 8

Why is thermostable bacterial DNA used

Answer 8

Thermostable DNA polymerase, such as Taq polymerase, is crucial in PCR because it can withstand the high temperatures required for DNA denaturation (95°C). This stability allows the enzyme to remain active throughout the repeated heating and cooling cycles of PCR without denaturing.

Question 9

What role does Mg²⁺ play in the PCR reaction?

Answer 9

Mg²⁺ is a cofactor that is essential for the activity of DNA polymerase in PCR. It stabilizes the negative charges on the DNA backbone and the dNTPs, ensuring proper function of the polymerase and efficient DNA synthesis.

Question 10

What are the primers used for

Answer 10

To target particular parts of the genome

Question 12

Why is it necessary to know the nucleotide sequence of the target DNA before performing PCR?

Answer 12

The nucleotide sequence must be known to design specific primers that will bind to the beginning and end of the target DNA sequence, ensuring that only the desired region is amplified.

Question 13

Describe the steps involved in the PCR process.

Answer 13

• Denaturation (95°C): DNA strands are separated.
• Annealing (~55°C): Primers bind to the target DNA sequence.
• Extension (72°C): DNA polymerase synthesizes the new DNA strand.
  This process repeats for 25-35 cycles, each cycle doubling the amount of target DNA.

Question 14

How can PCR products be visualized and analyzed?

Answer 14

• Gel electrophoresis(product size)
• Sanger sequencing(arrangement of bases in the PCR product)

Question 15

What are the limitations of using gel electrophoresis for analyzing PCR products?

Answer 15

Gel electrophoresis only provides information about the size of the PCR product, not its sequence. Additionally, if the primer design is not specific enough, non-specific amplification products may be produced, which can complicate the interpretation of results.

Question 16

What is array hybridisation

Answer 16

It is a massively parallel technique used to genotype multiple specific germline SNPs in a sample

Question 17

Why is array hybridisation important?

Answer 17

It can tell
us the germline genotype of an individual
across hundreds of thousands of different SNPs.

Question 18

What distinguishes germline SNPs from somatic mutations in array hybridization?

Answer 18

Germline SNPs are inherited genetic variations present in every cell of an individual and can be detected by SNP arrays. Somatic mutations, on the other hand, occur in specific cells during a person’s life and are not inherited; they cannot be detected by SNP arrays designed to detect germline variations.

Question 19

What are some clinical applications of SNP arrays?

Answer 19

• SNP arrays are used to identify carriers of known single-gene disorders
• to research genetic risk factors for complex traits,
• to diagnose specific genetic conditions.

Question 20

How does array hybridisation work for SNP genotyping?

Answer 20

1. Oligonucleotides(100,00 and 50bp in length) on the array are designed to target specific SNPs, with each oligonucleotide stopping one base before the SNP.
2. The oligonucleotides are bound to tiny silica
   beads(withs its own primer attached) spread across the microarray.
3. DNA fragments are added to the array with fluorescently tagged nucleotides.
4. DNA hybridizes to the oligonucleotides, and a single base extension occurs.
5. The genotype is read based on the color emitted after laser excitation. The specific base added is identified by its fluorescent color, allowing for the determination of the genotype at each SNP.

Question 21

What is Array-based Comparative Genomic Hybridisation (aCGH)?

Answer 21

A technique used to detect aneuploidy and chromosomal copy number variations (CNVs) on a genome-wide scale.

Question 22

Why is aCGH important

Answer 22

It is a rapid and cost-effective method for detecting genetic changes that result in DNA gain or loss.

Question 23

What are the steps involved in conducting aCGH?

Answer 23

1. Isolate genomic DNA from the patient and reference samples.
2. Digest the DNA samples.
3. Label patient DNA with one fluorescent dye and reference DNA with another.
4. Hybridize both samples to a microarray.
5. Wash the microarray post-hybridization.
6. Scan the array and analyze the data to detect duplications or deletions.

Question 24

What types of genetic variations can aCGH detect?

Answer 24

aCGH can detect aneuploidy, chromosomal imbalances, and CNVs as small as 50 kb.

Question 25

Why is it necessary to use both patient and reference DNA in aCGH?

Answer 25

Using both patient and reference DNA in aCGH allows for a comparative analysis. The two DNA samples are competitively hybridized to the same array, and differences in fluorescence ratios reveal duplications or deletions in the patient’s DNA relative to the reference.

Question 26

How does aCGH detect duplications and deletions in the genome?

Answer 26

if the patient’s DNA has more copies of a region (duplication), it will produce a higher signal relative to the reference DNA. If the patient’s DNA has fewer copies (deletion), the signal will be lower compared to the reference, allowing these variations to be detected.

Question 27

What are the clinical applications of aCGH?

Answer 27

• Diagnosing Down syndrome (extra copy of chromosome 21)
• Detecting genetic changes in cancer, such as tumor suppressor gene loss or oncogene amplification
• Diagnosing unexplained developmental delays, intellectual disabilities, autism spectrum disorders, and congenital anomalies

Question 28

What are the strengths of aCGH

Answer 28

Can detect small CNVs (as small as 50 kb), is cheap, less labor-intensive, and provides whole-genome coverage.