Ch. 19 Part 2

Question 1

What are the components of Sanger sequencing

Answer 1

template DNA, Taq polymerase, dNTPs, ddNTPs, primers, buffers

Question 2

Why are ddNTPs used in Sanger sequencing

Answer 2

because they terminate at a specific nucleotide (no 3’ OH)

Question 3

How many primers are used in Sanger sequencing?

Answer 3

one (linear amplification)

Question 4

When looking at a gel with a Sanger sequence- which way do you read it?

Answer 4

bottom to top across all 4 lanes

Question 5

What is a Genomic Library

Answer 5

multiple copies of DNA are digested (restriction endonucleases); produces overlapping DNA fragments; each fragment is joined to a cloning vector and transferred to bacterial cells; produces a set of clones containing overlapping genomic fragments (some containing the gene of interest)

Question 6

What occurs during the library screening?

Answer 6

bacterial colonies with vectors containing genomic DNA, RNA, or proteins are spotted onto nylon membrane; membranes are screened for sequences of interest through hybridization of radiolabeled probe

Question 7

Fluoresence in situ hybridization

Answer 7

using fluorescently labeled DNA probes we can identify cellular or chromosomal location of a sequence of interest

Question 8

Positional Cloning

Answer 8

we can find a gene of interest using linkage analysis with other mapped genes

Question 9

The Northern Blot Analysis

Answer 9

Evaluates RNA expression in various tissues, RNA is transferred from a gel to a membrane and is then probed with a radioactively labeled sequence complementary to the gene we are investigating

Question 10

Microarrays

Answer 10

RNA expression analysis; allows the simultaneous evaluation of expression levels for hundreds or thousands loci

Question 11

Isolating mRNA to make a cDNA library

Answer 11

uses oligo-dT to isolate mRNA (contains a poly-T tail which binds to mRNA’s poly-A tail) (Poly-A selection)

Question 12

We can use the relative abundance of cDNA to represent the relative abundance of ____

Answer 12

RNA in the cell

Question 13

What are the steps in next-gen sequencing

Answer 13

fragment sample (randomly sheer), ligate adapters (adapters provide a known sequences for primers to bind), isolate fragments and amplify, and synthesize DNA with primers targeting adapter sequence

Question 14

What kinds of sequencing made the $1000 genome possible

Answer 14

next gen and 3rd gen sequencing

Question 15

In next gen sequencing, millions of short sequencing reads are assembled into _____ based on sequence similarity

Answer 15

contigs

Question 16

RNA sequencing

Answer 16

a way to study gene expression by determining the presence of RnA molecules in a cell by sequencing cDNAs from RNA

Question 17

A contig is

Answer 17

a set of overlapping fragments that form a continuous stretch of DNA

Question 18

What is the basis of chromosome walking

Answer 18

the fact that a genomic library consists of a set of overlapping DNA fragments- we start with a cloned gene marker that is close to the new gene of interest so that the “walk” will be as short as possible

Question 19

Southern blotting investigates…., Northern blotting investigates…., Western blotting investigates….

Answer 19

DNA, RNA, proteins

Question 20

How can we determine how much gene expression there is?

Answer 20

through RNA expression analysis