DNA 2 Flashcards by Mohammad Zain

Define sequence-tagged site (STS)

short region along the genome (200 to 300 bases long) whose exact sequence is found nowhere else in the genome

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What is the significance of identifying a sequence-tagged site (STS)?

it is an effective method for physical mapping of the genome

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What is a commonly used sequence-tagged site (STS)?

Expressed Sequence Tag (EST)

–> unique and is produced by partial sequencing of cDNA library clones

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What is a Expressed Sequence Tag (EST)?

short DNA sequence (200–800 bp) derived from mRNA into cDNA

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What info do ESTs provide?

about genes that are actively transcribed in the sampled tissue or cell type

–> gene expression

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What is ‘physical map’ for?

shows the precise physical distances between genes or markers in terms of base pairs (bp)

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What is cytogenetic map for?

visual appearance of chromosomes under a microscope, including the location of genes or markers relative to chromosomal bands.

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What is genetic map for?

shows the relative positions of genes or genetic markers

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Define pharmacogenetics

study of how variations in a single gene (or a small number of genes) affect an individual’s response to a specific drug

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Define pharmacogenomics

broader study of how an individual’s entire genome influences their response to drugs.

–> focus on genome wide association studies (GWAS)

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What is the key difference between pharmacogenomics and pharmacogenetics?

pharmacogenetics: focuses on single genes or a small number of genes.

pharmacogenomics: Explores the entire genome and its influence on drug response

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What is HapMap?

creating a comprehensive map of human genetic variation by identifying and cataloging common patterns of genetic differences, known as haplotypes, across different populations

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Define haplotype

A haplotype is a group of DNA variations (or polymorphisms) that are inherited together on the same chromosome.

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What are the different studies used to study genes and genetic variations?

SNP arrays
Array CGH
NGS

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Define SNP

locations within the human genome where the type of nucleotide present (A,T,G, or C) can differ between individuals

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What is the full form of Array CGH?

CGH = Comparative genomic hybridization

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What is the purpose of array CGH?

compares patient’s genome against reference genome and identifies differences

–> locates regions of genomic imbalances in the
patient.

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Describe the technique of Array CGH

DNA chips coated with ssDNA probe
DNA/mRNA extracted from samples
samples labeled with diff fluorescent dye
hybridization
detection

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What are the challenges associated with DNA sequencing?

Variability in sample quality

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For DNA sequencing, where does the variability in sample quality arise from?

Formalin-Fixed Paraffin-Embedded (FFPE) Samples can damage DNA by crosslinks, breaks, accumulation of ssDNA and damages

–> special care when preparing long-read libraries for single molecule methods

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How big is the haploid human genome (in base pairs)?

3 billion

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Why is the HapMap project important?

provides tag SNPs, thus one can perform the genotype study with fewer SNPs
helps in finding the target gene

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How does HapMap provide tag SNPs?

certain SNPs are inherited together as a group so

e.g. for a gene with 20 SNPs that extend across
6,000 bases of DNA, genotyping 3 tag SNPs out of the 20 SNPs is sufficient to identify these haplotypes uniquely

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How many samples were used HapMap phase 1 project?

Study These Flashcards

270 samples
1 mil SNPs typed

How many SNPs were characterized in phase II of HapMap project?

over 3.1 million from 270 samples

What are the groups that participated in HapMap project?

Japan UK Canada China US

How many DNA samples were used HapMap Project Phase III?

1397

What are the challenges in GWAS associations?

1. which is causal variant? 2. which cell type the variants act? 3.. which genes regulated by the variants?

How many genes have been identified with proven or potential role in HIV therapy?

126

What are the major challenges of disease gene discovery?

1."unequivocal genotype" or even an "unequivocal phenotype" is virtually impossible to achieve in current limited-size studies of human populations 2. CNV not easy to define by short reads

What is copy number variant (CNV)?

large insertion/deletion or segmental duplication polymorphisms (SDs) in the human genome

Why is the study of CNV important?

CNVs can affect: -Gene dosage (number of functional gene copies). -Regulation of nearby genes. -Chromosomal stability.

What is RNAi (RNA interference technology) for?

1. determine the function of genes 2. assist in the development of new drugs

In what diseases do siRNAs prove to have therapeutic value?

diseases that are the result of alterations in gene activity

What is the characteristic of siRNA?

- dsRNA - 20-24 bp (normally 21) - operates within RNA interference pathway - degrades mRNA w/ complementary sequence --> prevent translation

What is the mechanim of siRNA?

1. endo-ribonuclease Dicer cuts dsRNA into siRNA 2. siRNA enter cell & incroporated into other proteins (eg AGO) to form RISC 3. siRNA unwinds to from ss siRNA 4. ss si RNA (part of RISC) binds to target mRNA 5. mRNA cleavage and mRNA is cut 6. mRNA recognized as abnormal by cell and degraded 7. no translation

What is the shortcoming of siRNA? How is it overcome?

effect is only transient, esp in rapidly dividing cells sol--> expression vector for the siRNA: shRNA

How is shRNA structurally different from siRNA?

shRNA has a short loop between the two strands

State a major difference between shRNA and siRNA

1. shRNA is delivered as a DNA vector whereas siRNA is delivered as RNA oligonucleotides 2. shRNA produced endogenously

What is microRNA (miRNA)?

1. ss non-coding RNA 2. 22 nucleotides 3. found in plants, animals and viruses 4. derived from RNA trabscipts that fold back on themselves to from short hairpin 5. can be derived from an intron

Describe how miRNA mediates gene silencing

M1) on initiation process, prevent assembling of initiation complex ir recruiting of 40S subunit M2) on ribosome assembly M3) on translation process M7,M8) degrade mRNA

Can shRNA and siRNA also carry out translation inhibition (M1, M2 and M3) just like miRNA?

rare but possible shRNA and siRNA's primary action is mRNA cleavage whereas miRNA primary action is translation inhibition

What are some biomolecules involved with the biogenesis of miRNA? rank in order

1. DROSHA 2. DRGC8 (Pasha) 3. DICER 4. AGO

What is the role of DOSHA in the biogenesis of miRNA?

- is class 2 ribonuclease III enzyme - initiation step of miRNA processing in the nucleus

What is the role of DRGC8 (pasha) in the biogenesis of miRNA?

- RNA binding protein - binds ss fragments of the pre-miRNA that are required for proper processing

What is the role of DICER in the biogenesis of miRNA?

- is RNase - further process pre-miRNA into mature miRNAs

What is the role of AGO in the biogenesis of miRNA?

- small RNAs - small RNAs guide Argonaute (AGO) proteins to their specific targets through base pairing, which then leads to mRNA

What is the full form of CRISPR?

Clustered Regularly Interspaced Short Palindromic Repeat

What is Cas9?

an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence

Which bacteria is CRISPR from?

Streptococcus pyogenes --> mild superficial skin infections to life-threatening systemic diseases or release toxins that lead to scarlet fever

What are the steps in CRISPR Cas9 gene editing?

1. Designing the Guide RNA (gRNA) with 20 nucleotide sequence complementary to DNA sequence 2. gRNA introduce into cell along with Cas9 3. Cas9 bind to gRNA 4. target recognition 5. Cas9 introduce double strand break (DNA cleavage) 6. DNA repair

What are the 2 kinds of DNA repair in CRISPR Cas9 in gene editing?

1. Non-Homologous End Joining (NHEJ) 2. Homology-Directed Repair (HDR)

Describe Non-Homologous End Joining (NHEJ) in CRISPR Cas9 gene editing

broken ends joing --> introduces small insertions or deletions (indels), which may disrupt the target gene

Describe Homology-Directed Repair (HDR) in CRISPR Cas9 gene editing

the cell uses new DNA template to repair the break, allowing for precise insertion, deletion, or replacement of DNA --> correct mutations

Define 'off-target effects of Cas9'

unintended cleavage and mutations at untargeted genomic sites showing a similar but not an identical sequence compared to the target site

What is somatic gene editing?

genetic interventions that will not pass to offspring --> allowed in humans

What is germinal gene editing?

genetic interventions that will be transmitted to progeny --> allowed in non-humans

DNA 2 Flashcards

(58 cards)