Recombinant DNA

Question 1

DNA polymorphism

Answer 1

refers to differences in the DNA sequence btwn individuals

Question 2

recombinant DNA

Answer 2

recombining different DNAs to form a new DNA strand

Question 3

What are the 2 enzymes needed to make recombinant DNA?

Answer 3

restriction endonucleases and DNA ligase

Question 4

restriction endonuclease

Answer 4

enzyme that recognizes specific DNA sequences (usually palindromic, 4-8 bp long) and cuts the DNA

Question 5

DNA ligase

Answer 5

seals DNA strands when they are close together (sticky ends lines up)

Question 6

Cloning vector

Answer 6

mechanism to carry foreign DNA into a cell, can be a plasmid or a virus

Question 7

Requirements of Cloning Vector (5)

Answer 7

1. autonomously replicate 2. separable from host DNA 3. unique restriction sites for inserting forgein DNA 4. can carry foreign DNA 5. must have a selection mechanism

Question 8

Key features of a plasmid (5)

Answer 8

1. origin of replication 2. many unique restriction sites 3. can carry up to 10kb DNA 4. antibiotic resistance genes 5. circular double strand of DNA

Question 9

2 types of DNA clones

Answer 9

1. genomic DNA clones 2. cDNA clone

Question 10

genomic DNA clone

Answer 10

1. represents a segment of genomic DNA (includes introns/exons/promoters/enhancers and junk DNA) 2. can be isolated from any tissue

Question 11

How big is the human genome?

Answer 11

3 billion bp

Question 12

How big is an average gene?

Answer 12

> 30kb

Question 13

cDNA clone

Answer 13

1. prepared by copying mRNA sequence into DNA (NO introns, promoters, enhancers) 2. contains exons 3. must be selective about what tissue it comes from bc not all genes are transcribed in all tissues

Question 14

What do you need to turn mRNA into cDNA?

Answer 14

use poly T primer for reverse transcriptase to make DNA, then synthesize 2nd strand with DNA polymerase to yield double stranded cDNA

Question 15

What drives DNA across an agarose gel during electrophoresis?

Answer 15

stonr negative charge of DNA, small pieces migrate faster

Question 16

Southern Blot

Answer 16

separate segments of DNA on agarose gel, then label the segment of interest using a radioactive complementary probe, develop X-ray to see which fragment is the one of interest

Question 17

How do you make a probe?

Answer 17

1. denature double stranded DNA 2. add DNA pol, primers and radioactive dNTPs 3. strands will be synthesized that are complementary and labeled

Question 18

Restriction Fragment Length Polymorphism

Answer 18

when a polymorphism alters the restriction endonucleases ability to cut a site and chnages the length of the restriction fragment (variations in DNA between chromosomes or individuals to track inheritance patterns)

Question 19

Purpose of PCR

Answer 19

amplify a small quantity of DNA

Question 20

Components of PCR (5)

Answer 20

1. sample of DNA 2. complementary oligonucleotide primers 3. nucleotides 4. thermal-stable DNA polymerase 5. pH buffer and salts

Question 21

microsatellite repeats

Answer 21

repeats of di- or tri-nucleotides whose number of copies will fluctuate among individuals

Question 22

How does PCR help determine DNA polymorphisms?

Answer 22

by amplifying the microsatellite repeats you can see how long they are, which will be unique to an individual

Question 23

single nucleotide polymorphisms

Answer 23

differences of one nucleotide between individuals that do not alter restriction sites (happens 1/1250 bp)

Question 24

2 techinques for sequencing DNA

Answer 24

1. sanger-dideoxy chain terminating method 2. parallel DNA sequencing (solexa)

Question 25

Sanger-dideoxy method

Answer 25

1. DNA polymerase inserts ddNTPs which end polymerization at that base 2. ddNTPs are added in low concentrations to make sure all the lengths of complementary DNA strand are made for each base (A,G,C,T) 3. each base that is fluorescently labelled uniquely 5. fluorescent detector reads the end bases of fragments increasing in size (5’ base of complementary strand is shortest/comes out first)

Question 26

parallel DNA sequencing (solexa)

Answer 26

way to sequence nucleis acid polymers side by side (75-100 bp sequences of up to 200million different DNA molecules)

Question 27

2 ways to measure gene expression

Answer 27

1. quantitative RT-PCR (for single sequence) 2. microarrays (for thousands of genes at once)

Question 28

Reverse Transcriptase PCR

Answer 28

amplifies mRNA (use poly T primer), reverse transcriptase makes cDNA which becomes the template for PCR reaction, perform 30 cycles and measure how quickly mRNA increases (more mRNA = faster amplification)

Question 29

Microarray Analyses

Answer 29

use cDNA collection to measure relative changes in RNA levels between two samples by looking at relative quantities of the labeled mRNA from the two samples binds to each gene specific cDNA oligonucleotide on the template (can see changes in the pattern of gene expression in the two samples)