Molecular Genetics (Unit 3) - chapter 8

Question 1

______ is the intentional production of new genes
and alteration of genomes by the substitution or introduction of new genetic material

Answer 1

Genetic engineering

Question 2

_______ Is a fragment of DNA composed of two sequences originating from at least two different sources

Answer 2

Recombinant DNA

Question 3

In order to form recombinant DNA, a molecular biologist
requires tools to ____, ____, and ____ DNA

Answer 3

cut, join, and replicate

Question 4

These tool include:

Answer 4

Restriction enzymes
DNA ligase
Plasmids

Question 5

An enzyme that cuts DNA at specific base pair sequence

Answer 5

Restriction Enzymes

Question 6

Each type of restriction enzyme is a able to recognize a
specific sequence of ______ that is known as its
_______ site

Answer 6

nucleotides
recognition

Question 6

A fragment that is produced when a DNA strand is cut by
these enzymes is called a ________

Answer 6

restriction fragment

Question 7

Restriction enzymes are
biological molecules, which are
isolated from _______

Answer 7

bacteria

Question 8

The function of these enzymes in bacteria is to:

Answer 8

cut foreign viral DNA into pieces

Question 9

Two types of cuts are possible

______ ends occur when cuts are
made straight across the DNA

_____ ends occurs when cuts are
made in a zigzag across the DNA

Answer 9

Blunt

Sticky

Question 10

_______ an enzyme that is used for “reassembly” of DNA
sticky ends

Answer 10

DNA ligase

Question 11

__ DNA ligase works well with blunt ends

Answer 11

T4

Question 12

A desired gene must be inserted into a _____ location

Answer 12

specific

Question 12

______ are small, circular pieces of DNA that are found in
bacteria

Answer 12

Plasmids

Question 13

DNA ligase Joins ______ bonds together

Answer 13

phosphodiester

Question 14

A plasmid that has been designed to transfer foreign genetic material into a cell is called a _____

Answer 14

vector

Question 15

The same restriction enzyme is used both on the source DNA
to __________ and the plasmid so that they will have
the same sticky ends.

Answer 15

isolate target DNA

Question 16

Cloning DNA fragments proccess diagram:

Question 17

Bacteria are able to uptake a plasmids under specific
conditions The introduction of DNA from another source is
known as ______

Answer 17

transformation

Question 17

_______ or commonly called plasmid maps are a
diagram that illustrates restriction enzyme recognition sites and the distances, measured in base pairs, between sites.

Answer 17

Restriction maps

Question 18

is a technique used to make a large number of copies of a DNA sequence.

Answer 18

Polymerase Chain Reaction (PCR)

Question 18

There are three steps to PCR:

Answer 18

Denaturation
Annealing
Elongation

Question 19

______- Cool the mixture to 50-65oC to allow primers to
anneal to the DNA

Answer 19

Annealing

Question 20

_______ – The DNA target sequence is heated to
94-96oC to denature it into single strands

Answer 20

Denaturation

Question 21

Millions of copies of the _________ can be produced in two hours.

Answer 21

DNA sequence

Question 21

________ – Heat to 72oC for the DNA polymerase to extend strand

Answer 21

Annealing

Question 21

The cycle is ______ several times. After the _____ cycle, two of the eight match target sequence.

Answer 21

repeated third

Question 22

A method for separating large molecules such as DNA, RNA, and proteins

Answer 22

Gel Electrophoresis

Question 22

Gel electrophoresis separates DNA fragments based on their _____ and ______ properties

Answer 22

chemical and physical

Question 22

DNA fragments migrate through the gel at a rate that is inversely proportional to their ___. The smaller the fragment is, the ______ it will travel through the gel.

Answer 22

size faster

Question 22

To be able to see the DNA fragments after the gel has been run, _______ is used to stain the gel.

Answer 22

ethidium bromide

Question 23

________ is a process in which the sequence of a strand of DNA is determined

Answer 23

DNA sequencing

Question 23

Uses of dna sequencing:

Answer 23

for understanding how a gene functions, mutations, locate regulatory sequences and compare homologous genes of different species

Question 23

understand Chain Termination (Sanger Method)

Answer 23

-Developed by Fredrick Sanger in 1970’s -It relies on the addition of a labelled dideoxynucleotide (ddNTP) to a growing DNA strand -ddNTPs prevent any further binding of nucleotides -The labels on the ddNTPs are dyes that fluoresce and can be used to identify the specific base when exposed to the laser light -Four reaction tubes are set up, each with many normal nucleotides (dATP, dTTP, dGTP, dCTP), multiple copies of the DNA to be sequenced, DNA polymerase, a DNA primer). -Only one of the labelled dideoxynucleotides (ddATP, ddTTTP, ddGTP, and ddCTP) are added each tube -This produces fragments of different sizes, each with their last nucleotide labelled

Question 24

Sequencing the Results:

Answer 24

The contents of the four tubes are combined and separated by gel electrophoresis The sequence can then be determined by reading the order of the labelling type that corresponds to a specific ddNTP This process is now computerized (fig 5 p.381)

Question 24

Whole Genome Shotgun method:

Answer 24

Involves producing many DNA fragments using a pressurized syringe Then uses Sanger sequencing and computers to sequence and reassemble the fragments (fig 6 p.381) Drawback of this method is for repeating segments- its hard to detect where these segments are repeated. This sometimes causes some errors in the results.

Question 24

Analyzing Genomes:

Answer 24

Once the DNA has been sequenced it can be analyzed for its actual sequence (structural genomics) and/or for its function (functional genomics) Most research is focused on functional genomics and it uses bioinformatics Bioinformatics uses both lab experimental data and computerized analysis

Question 25

Nanopore Sequencing:

Answer 25

Individual strands of DNA through microscopic holes called nanopores The sequence of the DNA is read as it passes through the pore by the unique current each bases conducts as it passes through the silicon sheet

Question 25

mRNA is isolated from a reference cell (normal) and an experimental cell (eg. cancer cell) Used to build complementary (cDNA) libraries cDNA sequences are labelled with fluorescent dyes Green for normal cDNA Red for experimental cDNA cDNA strands are denatured into single strands, then placed on microarray

Question 25

DNA microarrays:

Answer 25

Helps scientists pinpoint the functions of specific genes Microarray or gene chip, is designed to hold many individual DNA samples Samples are called probes – known sequences of genes for the organism/cell under study Location of each probe on the gene chip is also known

Question 26

cDNA will hybridize to any probes on the gene chip that they share a complementary sequence with Any non-complementary sequences will be washed off the chip Fluorescence is detected and analyzed Green spots mean normal cells express that gene Red spots mean experimental cells express that gene Yellow spots mean both cells express that gene :. can determine which genes have altered expression in the experimental cells Advantageous because thousands of genes can be analyzed at the same time