Gene Structure/fxn, Tools Molec Gene Analysis

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Phosphodiester bonds

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The way that adjacent nucleotides are linked in the 3'-5' direction

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What direction does DNA replication proceed?

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5' --> 3'

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Where is the origin of replication located?

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The site of initiation of DNA synthesis, or origin of replication, is located at intervals on each chromosome.

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Nucleosomes

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DNA complexed with histone proteins.

Basic structural unit of chromatin.

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Solenoid

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A helical, secondary chromatin structure.

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Intergenic DNA

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DNA that is not part of a gene. Function isn't clear.

About 25% is repetitive sequences, including both dispersed repetitive elements and satellite DNA.

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Promoter

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Region of a few hundred bps, located 5' of transcription start site, containing sequences involved in transcriptional regulation.

May contain TATA box, CCAAT box

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RNA processing

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1) Addition of Poly A tail
2) Removal of introns (splicing)
3) Splice junctions - sequences required for correct splicing
4) Untranslated regions

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Stop codons

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UGA, UAA, UAG

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Splice junction mutations

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1) Can destroy a splice junction (creating an additional sequence that's usually an intron)

2) Can create a new splice site (resulting in loss of sequences that are supposed to be translated)

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Mutations in regulatory region (eg. enhancer, promoter, etc.)

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Won't change the structure or biochemical properties of the protein, but can significantly affect how much protein will get made.

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Gene mapping

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determine on which chromosome and at what location on that chromosome a particular gene resides.

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Linkage analysis

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Technique used to measure how close two genes are to each other in the genome.

Genes that are close together tend to segregate together in meiosis because it's less likely recombination will occur.

Genes close together are said to be tightly linked.

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Genetic marker

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stretch of DNA for which the map location has been id'd by linkage analysis. Used as a reference point in mapping genes.

May be within a gene or in intergenic sequence.

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Polymorphic marker

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a marker with multiple, non-deleterious alleles.

Must occur at a frequency of at least 1%.

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Types of polymorphic markers

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RFLP
VNTR
microsatellite
SNP

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Gene therapy

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Adding a normal, non-disease gene to cells in the body that do not have a normal gene

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Direct genetic test

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Diagnostic test to determine whether a particular known mutation is present in the genes of an individual

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Indirect genetic test

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Diagnostic test to determine whether an invididual has inherited a chromosomal region containing a mutated gene. Uses polymorphic markers tightly linked to the disease gene.

Determines which alleles of a polymorphic marker the individual inherited and compares to family members, including affected individuals.

If the chromosomal region inherited by the individual can be distinguished/compared to family members' markers, then it can be determined whether the disease has been inherited.

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Molecular cloning

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1) Cut with restriction enzyme to generate restriction fragments

2) Get the fragments into bacterial or yeast vector

3) Vector replicates and produces clones of the sequences.

4) Isolate the plasmid DNA in large amounts.

Different types of vectors can hold different sized DNA fragments.

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Polymerase chain reaction

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Like molecular cloning, it amplifies a particular piece of DNA but it is a) quicker and b) able to amplify only smaller amounts of DNA (few kilobases max)

1) Denature DNA w heat
2) Anneal two oligonucleotide primers
3) Add special heat-resistant polymerase to add to these sequences, extending them to make full DNA strand.
4) Repeat many times to get huge amplification
5) Visualize by gel electrophoresis

<b>Must know some DNA sequence info in order to make the primers.</b>

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Advantages of PCR

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1) Fast
2) Automatic (in a machine)
3) Don't need a whole lot of DNA

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Disadvantages/potential problems of PCR

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1) Annealing/priming may be unspecific
2) Contamination; must do negative control experiments
3) Product size is limited (often need molecular cloning to amplify an entire gene in one segment.)

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Southern blot

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1) Restriction enzyme digestion of DNA
2) Gel electrophoresis
3) Transfer of DNA onto filter paper (The Blotting)
4) Hybridization with a DNA probe
5) Exposure to X-ray film to reveal size of DNA fragment

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Northern blot

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RNA is run on a gel and probed with DNA.

No restriction enzyme use.

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Uses of Southern blot

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1) Detect gene deletions >100 bp (as in for genetic diseases. Smaller deletions can be detected with PCR)
2) RFLP analysis
3) To determine number of tandem repeats (dx for Huntingtons)
4) paternity testing

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Example of uses for Northern blots

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Pattern of expression of candidate gene for genetic disease

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DNA sequencing

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Getting easier as technology improves.

May replace other methods in future.

Used to detect SNPs, which usually have two alleles.

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Allele-Specific Oligonucleotides (ASO)

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Direct test for detecting mutations of one or a few bases.

The sequence of the mutation AND the normal sequence must be known.
1) Obtain DNA
2) Amplify part of the gene of interest with PCR and Label the DNA
3) Hybridize amplified and labeled patient DNA to filter paper with <b>allele specific oligonucleotides</b> immobilized on it, using stringent conditions (only a perfect match will hybridize).

4) Determine whether patient DNA hybridizes to normal or mutant sequence, or both (heterozygous).

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What is cDNA? How do you make it?

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cDNA is complementary DNA to mRNA.

You make it by using mRNA as a template in a reaction which uses a primer, nucleotides, and reverse transcriptase.

Then you get an mRNA-DNA hybrid.

Destroy the mRNA, add DNA polymerase and nucleotides.

The result is a collection of dsDNA fragments representing all the mRNA in a cell (can determine what proteins are getting expressed.)

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Microarray analysis

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Purpose: allow simultaneous hybridization to many genes (potentially ALL genes) such that each gene can be separately visualized.

Technique: Oligonucleotide probes representing the genes are generated, attached to a very small chip. Samples representing the DNA or RNA from a person can be hybridized to the microarray.

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Gene expression arrays

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mRNA from selected cells is made into cDNA, labeled with a fluorescent tag, and hybridized to the oligonucleotides on the chip. Signal is detected with a laser scanner. Results in a profile of the genes that are expressed in the selected cells, and is quantitative, allowing comparisons between different cell samples. (eg. darker boxes would mean a particular gene is getting expressed more in an mRNA from a certain tissue than another.)

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Uses of microarrays in classification of different types of cancer

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Used to derive transcriptional profiles of 50-100 genes that help distinguish disease categories or make predictions.

based on idea that certain cancers express certain genes more.

Able to differentiate between acute myeloid and acute lymphoblastoid leukemia, for example.

<b>Soon, microarrays may be able to help determine a particular patient's response to a specific drug.</b>

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Pharmacogenetics

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Using an individual's genotype to determine what medications they'll respond to best.

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What is the approximate lifetime incidence of breast cancer in women?

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12% ; 1 in 8

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BRCA1

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Gene mutated in 1:500-800 women (1 in 40 in Ashkenazi Jews); confers a lifetime risk of 50-85% of breast cancer.

Inherited in autosomal dominant manner.

Men with the mutation have increased risk of breast cancer too.

Normal function: tumor suppressor genes and are involved in DNA repair of double-strand breaks

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BRCA2

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Gene mutated in about 1:500 women, conferring breast cancer risks similar to BRCA1, also inherited in autosomal dominant way.

Normal function: tumor suppressor genes and are involved in DNA repair of double-strand breaks

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What other cancers are carriers of BRCA1 and 2 mutations at risk for?

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ovarian, pancreatic, early-onset prostate, melanoma, colon cancer.

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American Cancer Society breast cancer screening guidelines

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1) Yearly mammograms above age 40.

2) Clinical breast exam every 3 years between 20-39, then every year for 40+

3) Women should report any breast changes to doctor at any age.

4) Women at increased risk should talk with doctors about benefits and limitations of starting mammograms earlier.

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Relative Risk

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incidence among exposed(with factor) / incidence rate on unexposed (without factor)

RR <1 : exposure is protective
RR = 1 : exposure is unrelated
RR >1 : exposure increases risk

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Odds Ratio

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An estimate of relative risk.

Odds of exposure among cases / Odds of exposure among controls

OR is particularly useful when prospective studies are not available. Frequently used in GWAS.

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What types of genomic changes are genome-wide association studies typically looking for?

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SNPs