1.4 - Major advances in genomics

Question 1

DNA sequencing

Answer 1

used to determine the exact order of the bases in a strand of DNA. Can be done by detecting how the bases are added to figure out the template DNA

Question 2

Sanger’s method

Answer 2

addition of dNTPs (the base units) extends the DNA chain, but the addition of ddNTPs cause the termination of strand elongation

Question 3

Pros and cons of Sanger’s method

Answer 3

Pros:
- Relatively accurate
Sequencers are cheaper

Cons:
- Can only be used for short - - DNA strands of 100 to 1000 base pairs
Labour intensive and time consuming

Question 4

Shotgun method

Answer 4

Long DNA is randomly broken into numerous small segments and are sequenced using Sanger’s method to obtain reads

Question 5

Pros and cons of Shotgun method

Answer 5

Pros:
- Fast and relatively accurate
- Allows the sequencing of multiple DNA fragments parellelly

Cons:
- The assembly part is computing-intesive and it’s difficult without an existing genome to match to

Question 6

Second generation sequencing: Next Generation Sequencing (NGS)

Answer 6

based on sequencing by synthesis - chain elongation without termination

An example is the Illumina method

Question 7

Illumina method

Answer 7

The DNA is cut into millions of small fragments. The adapters are added to each end, then the sequencing library is prepared

Question 8

Pros and cons of the Illumina method

Answer 8

Pros:
- High throughput
- Cost efficient

Cons:
- Relatively short read lengths (150-300bp)
- Large initial investment
- Problematic for long repetitive sequences at the assembly stage

Question 9

De novo sequencing

Answer 9

the initial sequencing of an organism. It requires large computing capacity to assemble the short DNA reads into a complete sequence. It serves as a reference sequence

Question 10

Resequencing

Answer 10

sequencing an individual of a species where a reference genome sequence is available. Faster and easier than de novo sequencing. Used to find variation among individuals

DOES NOT HAVE AN ASSEMBLY STEP. It’s replaced by mapping the fragments onto the reference genome

Question 11

Annotation

Answer 11

Process of attaching biological information to sequences. It follows the steps of:

1. Identifying non-coding regions
2. Identifying genes
3. Attaching biological function to the genes

Question 12

Transcriptome

Answer 12

the set of all RNA transcripts in an individual or a population of cells. Can be used to refer to all RNAs, or just mRNA (depends on context)

Question 13

2 methods of gene expression detection

Answer 13

1. RNA sequencing (but RNAs are not stable and difficult to work with)
2. Complementary DNA method (cDNA) - made using mRNA as template. All the cDNAs are sequenced using NGS

Question 14

RT-PCR (Reverse transcription polymerase chain reaction)

Answer 14

it combines reverse transcription of RNA into DNA (cDNA) and PCR to measure the amount of a specific RNA

Fluorescence is used in qPCR (real-time PCR) to monitor the amplification reaction

Question 15

Genetic marker

Answer 15

any altercation in a sequence of DNA or other genetic trait (gene product) that can be readily detected. They mainly consist of polymorphisms (various forms). Used to:

• Identify individuals, populations, or diseases
• Locate gene with known function
• Study genetic variation

Question 16

What are isozymes and allozymes

Answer 16

they are proteins having the same function but differ in size, shape, and the amount of charge. They can be separated via electrophoresis. They can be detected by staining

Question 17

Isozymes

Answer 17

different forms of the same enzyme that are encoded by different genes

Question 18

Allozymes

Answer 18

different forms of the same enzyme that are encoded by different alleles of the same gene

Question 19

Restriction fragment length polymorphism (RFLP)

Answer 19

cuts genomic DNA into pieces by restriction enzymes and separates the DNA segments. It helps detect genetic variation, but there are a limited number of markers

Question 20

Simple sequence repeats (SSRs)

Answer 20

aka microsatellites. The repeats are distributed at thousands of locations over the genome. It has high mutation rates, which may change the length of the repeats - genetic variation
3

Question 21

Single-nucleotide polymorphism (SNP)

Answer 21

SNP represents a difference in a single nucleotide of a DNA sequence

Question 22

Genome-wise association study (GWAS)

Answer 22

used to identify SNPs across genomes that are statistically associated with a particular trait. Needs large pool for SNP and phenotypic data

Question 23

Genome-environment association (GEA)

Answer 23

studies the relationship between each SNP and environmental variables. This is more indirect, but it can find stronger relationships than GWAS