Ch 1.2

Question 1

Screen libraries using probe

Answer 1

Use molecular marker closest to gene of interest to make a probe to screen a library. Isolate from library and use a region of its DNA to probe the library again. Screen libraries—>probe—>RE then gel elcto—>new probe, screen again

Question 2

Chromosomal Walking

Answer 2

a contig: set of overlapping clones

Question 3

Southern vs. Northern Blotting

Answer 3

Southern Blotting: A DNA sample run. [gel—>nitrocellulose gel—>hybridize with probe—>autoradioram] Northern blotting: An RNA sample is run in a gel and probed. [Useful in studies of gene expression:]

Question 4

Example of an environmental effect

Answer 4

Phenylketonuria (PKU) Early diagnosis of a new born allows control of diet (avoid phenylalanine) and prevents mental retardation.

Question 5

The polymerase chain reaction (PCR) What is it, what’s it do?

Answer 5

Allows the amplification of a selected DNA sequence from total DNA. Cloning without the use of cell systems. The procedure can only be applied when a short sequence of DNA is known on each side of the genomic region (gene) of interest (primers). The procedure involves three steps repeated many times.

Question 6

3 PCR steps

Answer 6

Sample: dsDNA + dNTPs + primers + Taq polymerase. Step 1: Denature dsDNA (heat at 95°C). Step 2: Annealing. Bind ssPRIMERS (cool to 48 - 60°C) Step 3: DNA Taq polymerase (heat stable) extends strands by incorporating free nucleotides (dNTPs) (72 °C).

Question 7

DNA sequence from single strand

Answer 7

Use a labeled primer (e.g. radioactive). DNA polymerase will extend sequence from primer by adding dNTPS.

Question 8

Sanger method

Answer 8

Run four reactions with addition of different ddNTPs (can not form phosphodiester bond with the next nucleotide to be incorporated  termination of elongation). Each reaction contains various truncated labeled DNA fragments that can be separated and identified by gel electrophoresis. Makes a sequence you can read thats complementary to DNA template strand

Question 9

Automated sequencing

Answer 9

A different fluorescent dye is used to label each of the four dideoxy terminators (see figure). The four reactions can now be run in one tube and run in a gel. Automated DNA sequencing machines can run up to 96 capillary electrophoresis simultaneously. 24hs. operation can yield over 100,000 nucleotides in a day (the human genome has three billion!)

Question 10

Shotgun

Answer 10

The target sequence (whole genome) is sheared into small fragments that are cloned. Templates are chosen at random and sequenced, yielding a ssDNA sequence (500-1000bp). If enough short random sequences are available (automated sequencing is crucial!), overlapping regions can be identified. The sequencing redundancy (fragments sequenced several times) aids in the assembly and increases sequence accuracy.

Question 11

Massive parallel sequencing 5 common things

Answer 11

1) Libraries are made by fragmentation of DNA and ligation to sequence adapters (No E. coli cell system!). 2) Amplification is done using beads or glass platforms (emPCR) 3) All sequencing reactions are run at the same time and with simultaneous detection (Massively parallel sequencing) 4) Shorter read length than with automated Sanger sequencing 5) Pair-end reads help in aligning to a reference genome

Question 12

Massive parallel: 3 types

Answer 12

Solid, Illumina, Pyrosequencing

Question 13

Pyrosequencing

Answer 13

It relies on the detection of pyrophosphate (PPi) release on nucleotide incorporation. PPi initiates a series of downstream reactions that ultimately produce light by the enzyme luciferase. The amount of light is proportional to the number of nucleotides incorporated. 1. signal realeased—>enzyme cascade—>light emission

Question 14

Illumina

Answer 14

DNA molecules are attached to primers on a slide and amplified (bridge amplification). Labeled nucleotides are then added, and non-incorporated nucleotides are washed away. A camera takes images of the fluorescently labeled nucleotides and the dye is chemically removed from the DNA, allowing a next cycle (attatch DNA to glass surface). Charac: glass surface and bridge amphilication

Question 15

Solid

Answer 15

Employs sequencing by ligation. The sequence of the DNA is determined by ligation of fluorescently labeled DNA probes using the DNA on the beads as a template. The preferential ligation by DNA ligase for matching sequences results in a signal informative of dinucleotide at that position. Every step is two at a time. Charc: ligation