Genetics: Exam 3

Genetic (linkage) maps vs. physical maps

Genetic (linkage) maps- approximate location of genes based on recombinant rates

Physical maps- more accurate; higher resolution than genetic maps

Describe shotgun sequencing.

Breaks up DNA into smaller fragments that are reassembled to reconstruct the original sequence

Short vs. long reads

Short reads- tend to have gaps in genome

Long reads- spans long variants; more comprehensive

2nd next-generation DNA sequencing

• a short read

• increased efficiency

  • a nucleotide is bound to a different fluorescent color which helps you to read the DNA sequence

3rd generation next-gen sequencing

• long read sequence

• There’s a nanopore method and PacBio Hifi method

Nanopore method

• has the longest reads

• reads nucleotides based on ELECTRICAL charge

PacBio Hifi method

• relies on sequencing a circular DNA molecule

• adds circular adapter to ends of DNA sequence

HiFi is more accurate than the nanopore method

List the key steps for a Tn-Seq

1) Select your transposon type

• Ex: bacteria, Drosophila, mice

2) Select your selectable marker

• Ex: antibiotic resistance gene, red eye color, and GFP

3) Clone transposon & selectable marker. Don’t forget the transposase!

4) Screen for transposition in your organism.

5) Expose transposed organisms to different conditions

6) Isolate genomic DNA from organisms grown in different conditions

7) Fragment genomic DNA

8) Ligate (combine) DNA fragments with a transposon specific adaptor

• Ex: transposon is designed to have restriction enzymes site (ex:Msel) allowing DNA to be digested and ligated to sequencing adaptor

9) Proceed with genome-wide DNA sequencing method

How to determine where the transposon inserted?

To find where the transposon inserted, you want to do a short read sequencing

You could also use a long read, if there are repetitive nucleotide sequences

How can you interpret the function of genes 1, 2, and 3 based on the number of reads?

(look at the image)

Sanger vs. next-gen sequencing

Sanger sequencing- only does 16 reactions per gel; lacks 3’ OH; adds fluorescently labeled ddNTPs

• Sanger sequencing is preferred, if you ONLY want to know the sequence of one or a few genes, instead of the whole genome!

Next-gen sequencing- 10,000 reactions per slide

What is genome wide association study (GWAS)?

• An approach that rapidly scans for markers, like SNPs, across the genome

• Once an marker (SNP) is identified, you can determine where it’s located and the DNA sequence associated with the trait

What is an SNP?

Site on genome where individual members of a species differ in a single base pair

Haplotype

Specific set of SNPs and other genetic variants observed on a single chromosome or part of chromosome

What if an SNP maps to a human gene of unknown function?

1) First, determine where the SNP is located in the gene.

• This can include a protein coding region (ORF), regulatory region (promoter/enhancer), intron (UTRs)

2) Search for similar genes in other species that have known function

Describe homologous genes, orthologs, & paralogs.

Homologous- genes that are evolutionarily related

Orthologs- homologous genes in different species

Paralogs- homologous genes arising by duplication in SAME organism

What are some mechanisms of genome evolution?

1) Mutation- causes the majority of genome evolution

2) Gene duplication- another source of genome evolution & gives rise to paralogs

3) Gene deletion

4) Exon shuffling- shuffling of genes through recombination

Synonymous mutations vs. nonsynonymous mutations

Synonymous mutations- do NOT alter amino acid sequence

• Ex: a silent mutation

Nonsynonymous mutations- ALTER amino acid sequence

Neutral-mutation hypothesis

Individuals with different molecular variants have EQUAL fitness

Balance hypothesis. Give an example

Genetic variations in populations that FAVOR variation

• Ex: overdominance- where the heterozygote has higher fitness than the homozygote

Define phylogeny and phylogenetic tree.

Phylogeny- evolutionary relationship among a group of organisms

Phylogenetic tree- a graphical representation of the evolutionary relationships among a group of organisms

Define transcriptomics. List single transcript and whole transcriptome methods.

Transcriptomics- measures all RNA molecules transcribed in genome

Single transcript: In-site hybridization (in-situ), northern blot, reverse transcription polymerase reaction (RT-PCR)

Whole transcriptome: cDNA microarray & RNA-seq

In-situ hybridization (ISH)

• Allow for precise localization of a specific segment of nucleic acids in a cell/tissue section

• RNA sequences are based on binding of a probe

• Probes are labeled with markers (ex: radioactive isotopes, fluorescence, etc.)

Advantage: helps resolve which cell type expression is present

Northern blot

• Begins with isolating RNA from cell, tissue, etc.

• Then, RNA gets ran on an agarose gel

• Uses electrical current to transfer RNA onto RNA-binding membrane

• Probe for whatever gene of interest

• If mRNA is present, probe will bind and you expose filter to film

  • The more intense the band is, the more the gene is expressed

Reverse transcription polymerase chain reaction (RT-PCR)

• Isolate RNA from cell/tissue/organism

• Reverse transcribe mRNA to complementary DNA (cDNA)

  • To do this, you need reverse transcriptase, dNTPs, & DNA primer

  • For eukaryotic cells, you need Oligo T (primer)

  • For prokaryotic cells, you need random hexamers (primer)

• Amplify gene of interest using PCR with gene specific primers, DNA polymerase & dNTPs

• Take amplified sequence and run it through gel electrophoresis to resolve RT-PCR DNA sequence

  • Intensity of band = expression of transcript (like in Northern blot)

DNA Microarray (also list its disadvantages)

It’s a chip that allows thousands of transcripts to be determined simultaneously

1) You need cells from 2 or more different conditions to isolate total RNA

2) Reverse transcribe mRNA to cDNA

3) Label different conditioned cells with fluorescent markers

4) cDNAs are mixed and hybridized to DNA probes on a chip

5) Then, the chip is scanned

• If there are equal amounts of expression in BOTH cell types of different conditions (yellow fluorescence)

• More expression of one cell types (ex: cancer cell)→ red fluorescence

• More expression of another cell type (ex: noncancer cell)→ green fluorescence

Disadvantages: low sensitivity, low dynamic range, no alternative splicing

RNA Sequencing (RNA-seq)

Allows you to identify ALL mRNA transcripts present in cell

1) Isolate total RNA from cells (specific whether prokaryotic or eukaryotic)

2) Select for type of RNA (in this case, mRNA)

3) Reverse transcribe RNA into cDNA using DNA primers, dNTPs, and reverse transcriptase

• Remember, for eukaryotic cells → Oligo T primer

• For prokaryotic cells→ Random hexamer primer

4) cDNA gets fragmented and adaptors are added

5) Lastly, sequence using short read sequencing for bacteria and long read sequencing for eukaryotes

Advantages: high sensitivity & high dynamic range

Why do you use short read sequencing for bacteria and long read sequencing for eukaryotes?

This is because long read sequencing allows alternatively spliced transcripts to be identified. Bacterial transcripts are not spliced.

Single cell vs. bulk RNA-seq

Involves the same steps as in RNA-seq; however….

• Single cell separates each INDIVIDUAL cell, while bulk keeps cells in a GROUP