18. SNPs & Human genetics

MIT OpenCourseWare

12 May 202048:33

EducationalLearning

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TLDRThe transcript describes a biology lecture explaining techniques for cloning and identifying human disease genes. It covers linkage mapping to find the chromosome location of a disease gene, constructing a physical map of cloned DNA fragments spanning the region, and walking along the chromosome to identify the causative gene. It explains making cDNA libraries to identify transcribed genes, and hybridization techniques like in situ hybridization to see if a gene is expressed in a specific tissue. Overall, it outlines key concepts and methods for positional cloning of human disease genes.

Takeaways

📖 The Sanger sequencing method is an elegant, though older, technique illustrating the concept of chain termination, important for understanding DNA sequencing fundamentals.
🕹️ Aniridia is a heritable eye disease lacking an iris, serving as an example for discussing gene cloning for disease understanding.
📈 Autosomal dominant inheritance is identified as the likely mode of inheritance for aniridia, based on family pedigree analysis.
🔬 Positional gene cloning involves identifying a gene based on its chromosomal location, using linkage analysis and molecular markers to narrow down the search.
📚 cDNA (complementary DNA) libraries are crucial for identifying genes expressed in specific tissues, as they are made from mRNA and lack introns, promoters, and other non-coding regions.
💎 The technique of chromosome walking helps in identifying and isolating genes related to specific diseases by sequentially cloning DNA fragments near known markers.
📡 Hybridization techniques, including in situ hybridization, allow for the identification of specific DNA sequences within tissues, enabling researchers to see where genes are expressed.
👨‍💻 The lecture emphasizes the importance of understanding foundational techniques like the Sanger method, even in the era of more advanced sequencing technologies.
🚨 The cloning of the aniridia gene (Pax6) illustrates the application of genetic techniques to understand and potentially treat genetic disorders.
📱 The concept of cDNA and its role in gene cloning and expression analysis highlights the bridge between gene expression and functional genomics.

Q & A

What is the disease called aniridia?
-Aniridia is a rare eye disease where the affected individual has an eye lacking an iris.
How is positional gene cloning used to identify disease genes?
-Positional gene cloning involves cloning a gene responsible for a disease based on its position in the genome, through steps like linkage mapping to identify the chromosome region and then constructing a physical map of overlapping DNA clones from that region.
What is complementary DNA (cDNA)?
-cDNA is DNA synthesized from mRNA via reverse transcription. It lacks introns and regulatory sequences present in genomic DNA.
How are cDNA libraries made?
-mRNA is purified using oligo dT beads, then converted to cDNA via reverse transcription. The cDNAs are cloned to create a library representing the genes expressed in the tissue.
How does a cDNA library differ from genomic DNA?
-A cDNA library lacks introns, promoters and other non-transcribed regions. It represents only the genes expressed in the source tissue.
What is chromosome walking?
-Chromosome walking is a technique to clone overlapping DNA fragments starting from an initial known sequence in order to cover an entire chromosome region of interest.
How is hybridization used to identify gene transcripts?
-Labeled DNA probes can hybridize/anneal to identical or similar sequences, allowing identification of clones containing a particular transcript without knowing the sequence a priori.
What does in situ hybridization show?
-In situ hybridization localizes specific mRNA transcripts within intact fixed tissues by hybridizing labeled probes, showing tissue-specific expression patterns.
How was the aniridia gene identified?
-The aniridia gene was identified by positional cloning, starting with linkage mapping and chromosome walking to clone the region, finding a homolog of the fly eyeless gene.
What happens when the eyeless gene is mis-expressed in flies?
-Ectopic expression of eyeless can induce formation of ectopic eyes on other body parts like legs, showing it is a master regulator of eye development.

Outlines

00:00

🧬 Introducing DNA Sequencing and the Sanger Method

This paragraph introduces DNA sequencing using the Sanger method, which involves four reactions with different dideoxynucleotides to generate a sequence ladder. A sequencing gel is passed around to demonstrate the fragment patterns corresponding to sequence.

05:02

👀 Describing the Eye Disease Aniridia

This paragraph describes aniridia, a rare inherited eye disease where the iris is missing. A pedigree analysis suggests it is autosomal dominant. The goal is to clone the aniridia gene using positional cloning.

10:07

🚶‍♂️ Linkage Mapping to Chromosome Walking

This paragraph outlines the process from linkage mapping to identify the chromosome region containing the aniridia gene, to chromosome walking using overlapping clones to span the region and identify genes.

15:08

🔎 Identifying Interesting Genes

This paragraph discusses criteria for identifying interesting genes during chromosome walking, including open reading frames, tissue-specific expression, and conserved genes with relevant functions.

20:08

📜 Creating cDNA Libraries to Identify Transcripts

This paragraph explains how to create cDNA libraries to identify transcribed sequences. cDNAs lack introns and regulatory regions but are proportional to expression levels across genes and tissues.

25:10

⬅️ Reverse Transcription to Make cDNA

This paragraph details the process of reverse transcription to make cDNA from mRNA using reverse transcriptase, going against the central dogma from RNA to DNA.

30:14

🧫 Purifying mRNA and Making cDNA

This paragraph walks through the steps to purify mRNA using its polyA tail, prime reverse transcription with oligo dT, degrade the RNA strand, and synthesize the second cDNA strand.

35:18

📚 Representation of Genes in cDNA Libraries

This paragraph explains that representation of genes in a cDNA library depends on expression levels. Unexpressed genes will not be present while highly expressed genes will have more clones.

40:23

💬 Using Hybridization to Detect Transcripts

This paragraph introduces DNA hybridization using labeled probes to detect sequence similarity, enabling identification of tissue-specific transcripts without knowing the sequence.

45:30

😯 Pax6 Master Eye Development Gene

This paragraph shows fly and human experiments demonstrating how the Pax6 gene is a master regular of eye development across species.

Mindmap

Keywords

💡DNA sequencing

DNA sequencing refers to methods for determining the order of nucleotides (A, T, C, G bases) within a DNA molecule. The video discusses Sanger sequencing, an older technique that uses chain termination with dideoxynucleotides. This is relevant for understanding how scientists historically were able to read DNA sequence to aid gene discovery.

💡positional cloning

Positional cloning refers to identifying the location of a gene on a chromosome through linkage analysis, then isolating the gene from that region. This is relevant as the video discusses strategies for cloning human disease genes.

💡chromosome walk

A chromosome walk is a technique used in positional cloning, where you start with a known DNA marker on a chromosome then progressively isolate overlapping DNA fragments to walk across the chromosome region. This allows you to span a gene location.

💡cDNA library

A cDNA (complementary DNA) library contains DNA copies of mRNA transcripts from a cell. It represents the genes that are actively expressed. This is relevant for identifying genes and transcripts present in specific cell types.

💡reverse transcriptase

Reverse transcriptase is a unique enzyme used by retroviruses to make DNA copies of their RNA genome. Molecular biologists exploit reverse transcriptase to make cDNA from mRNA via reverse transcription.

💡hybridization

Hybridization involves allowing labeled single-stranded DNA probes to hydrogen bond with complementary sequences, used to detect genes or transcripts without needing to know an exact sequence.

💡in situ hybridization

In situ hybridization allows you to visualize location of a specific mRNA transcript within a tissue sample via labeled probes. This reveals what cell types a gene is expressed in.

💡model organisms

Model organisms like fruit flies are used study human disease gene homologs and understand gene functions. The video discusses manipulating fly eyeless/Pax6 gene to better understand human aniridia.

💡aniridia

Aniridia is a rare inherited human eye disease caused by mutations in the PAX6 gene. It served as model for positional cloning of a disease gene in the video.

💡eyeless gene

The Drosophila eyeless gene is required for fly eye development. It is homologous to mammalian PAX6 gene. Its mis-expression in flies induces ectopic eye formation, illustrating PAX6's role in controlling eye development.

Highlights

Transcripts

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