DNA Technology and Pedigree

Reverse Transcriptase

Reverse Transcriptase

Converts RNA into cDNA, used if interested in amplifying RNA

Real-Time PCR

• SYBR green fluorescent dye

• Does not fluoresce much in presence of single-stranded DNA

• As PCR continues, you add more. It will glow more every cycle

Frederick Sanger (1918-2013)

• British scientist

• Developed widely used method for sequencing DNA from amplified fragments of PCR or cloning.

DNA sequencing 2

ddNTP is incorporated randomly, terminating DNA copy

DNA sequencing 3: What you need

1. Primer (oligonucleotide)

2. Template (what you want to sequence)

3. Normal dNTPs

4. Enzyme and buffer

ddNTPs

Stop extension randomly- millions of different sized fragments of every possible length result

Dye Sequencing 4

Fluorescent dyes are covalently bonded to ddNTP’s, and show up as different colors on a chromatogram

• Peak colors are different nucleotides in the sequence

• G- Yellow

• A- Green

• C- Blue

• T- Red

Pyrosequencing

• Recall when a dNTP is incorporated into a growing DNA strand, two covalently linked phosphates (pyrophosphate, PP1) are released.

• Machines detect light every time the phosphates are released and react w enzymes to form ATP and give off light.

Pyrosequencing Peak Height

• Double-height peak: Two nucleotides incorporated when precursor was added.

• Single-height peak: One nucleotide was incorporated when precursor was added.

• Absence when nucleotides are added means they could not be incorporated into new DNA. Template didn’t have complementary bases.

Site-specific mutagenesis

• In vitro targeting of specific cloned genes.

• Use PCR with mutant primers

Polymorphism

Quality or state of being able to assume different forms

Shape size color regardless of sex

DNA Polymorphism

• One or two more alternative forms of an allele from a chromosomal locus resulting from differences in DNA sequences or numbers of tandem repeats- Not necessarily a gene- can be anywhere.

DNA marker

A known DNA sequence, often of DNA polymorphisms- best ones are unique in the genome, STS (sequence-tagged sites).

SNPs are Common

• J. Craig Venter: Human Genome Project: 3.2 million SNPs in his diploid genome, arise from spontaneous mutations

• 1~ every 1 kbp (45% of genes are heterozygous)- about 13 million per person.

• Each gene is a STS- unique site in the genome

Southern Blotting

• DNA

• Analysis determines arrangement and location of restriction sites.

Northern Blotting

RNA

Western Blotting

Proteins

SNP’s

• Cause a single point mutation at a site- most common DNA polymorphism (95%)

• Harmful if in coding genes, or regulatory regions

Restriction Fragment Length Polymorphisms (RFLP’s)

• Restriction enzyme generated fragments of different lengths.

• Can be detected through southern blot or PCR.

Oligonucleotide Hybridization

• Short piece of RNA or DNA

Microarrays

• 1990s

• Ordered grid of probe single-stranded DNA molecules of known sequence (with many SNP combinations) fixed to a substance or microchip.

• Unlabeled probes (fixed to substrate) and free target DNA (from organism to be tested) with colored dyes.

• Yellow means both hybridized, other colors indicate degrees of hybridization.

• cDNA= DNA synthesized by reverse transcriptase from mRNA

STR

Population-level studies, relationship to individuals.

2-6 bp DNA sequences repeated tandemly up to about 100x in the genome.

High rate of mutation:

1. Non-coding regions of genome (not under selection)

2. Often mismatched during rep. Junk DNA

VNTR’s (Variable number tandem repeats)

• Similar to STR’s, but repeating unit is 7-20

• Larger length so southern blot.

• 1988: Exonerated someone in a murder trial. Golden State Killer 2018

DNA Fingerprinting

• Takes advantage of DNA polymorphisms to identify individuals for forensics.

• DNA from blood, RE’s isolated segment with marker, and VNTR probes used with Southern blot to obtain profile.

• Heterozygous

Gene therapy

Mostly for somatic cells to correct diseases present.

Disorders with single gene mutation for which a normal clone is available.

Procedure:

1. Take out cells

2. Introduce normal gene (transgene) with virus vectors into them (now a transgenic cell).

3. Replace in body

If therapy is successful, treatment is required because transgenic cells eventually die.

Pedigree

Pictorial representation of family history that outlines the inheritance of one or more characteristics.

Proband

Person for whom the pedigree is initiated; first affected person to come to the attention of a geneticist.

Autosomal Recessive Traits

• Affected people more commonly in matings among closely related people, known as consanguinity.

• Tend to skip generations.

Genetic Testing

Identifies mutations in key genes responsible for disease.

Carrier detection (heterozygote screening)

Genetic testing of adults to calculate probability of baby having disease- uses blood.

Cell-free (cfDNA) screening

Blood test of DNA fragments from placenta at 10 weeks- looks for chromosomal abnormalities and sex of baby

Prenatal diagnosis

Amniocentesis is done to perform genetic testing- now can be done on embryos before implantation.

Newborn screening

Testing newborn babies for genetic disease using blood samples.

Cons Genetic Testing

1. Gene for a disease hasn’t been found in the genome.

2. Gene has many mutations, making a single test unreliable.

3. The presence of gene does not always result in disease.

4. Many diseases are polygenic, or caused by multiple genes.