Genetics Ch 20: molecular technologies

Recombinant DNA

_____ technology can Isolate and manipulate DNA fragments to produce new arrangements

recombinant DNA molecules

gene cloning uses _______ are replicated to produce many identical copies of a gene

Gene cloning

_______ is the process of making many copies of a gene

Gene/DNA sequencing

_________ is important in the identification of alleles that cause cancer and inherited human diseases.

its cellular function.

1. The expression of a cloned gene can help to elucidate _______

active promoter

In Research, the coding sequence of a gene can be placed next to an ______and then introduced into a culture of cells that will express a large amount of the protein.

insulin

 Cloned genes can be introduced into bacteria to make pharmaceutical products such as _____.

transgenic

Cloned genes can be introduced into plants and animals to make ______ species with desirable traits.

chromosomal DNA

•Source of the gene or DNA segment of interest from the species that carries the gene

vector DNA

DNA that will replicate independently of the host chromosome/serves as the carrier of DNA segment to be cloned; originally derived from plasmids and viruses

host cell

cell that carries the vector

origin of replication

multiple cloning site

selectable marker

plasmids contain: (3)

plasmids

Most vectors are ______ – small circular pieces of DNA.

multiple cloning site

•Unique sites where pieces of DNA can be inserted on a plasmid

• Origin of replication

the ____ can be used by host cell enzymes to replicate the plasmid

selectable marker –

resistance gene that provides host cells with the ability to grow in the presence of a toxic substance; expression of the resistance gene selects for the growth of the host cells

 clone large pieces of DNA (alongside plasmids and viruses)

 cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs) can be used to …

restriction endonucleases or restriction enzymes

The enzymes used to cut or digest DNA are known as ______ or ______

restriction enzymes

these bind to specific DNA sequences and then cleave  the DNA at two defined locations, one on each strand

restriction enzymes

these are made by bacterial cells to protect them from invasion by foreign DNA

sticky ends

• •Certain types of restriction enzymes cut to generate “______ “ which can hydrogen bond to DNA from another source with complementary ends

DNA ligase

once sticky ends are hydrogen bonded to each other ____ is added to catalyze the formation of covalent bonds between the DNA backbones

palindromic

______ sequences are identical when read in the opposite direction in the complementary strand

palindromic

restruction enzymes usually recognize sequences that are _____

• 5′ G A A T T C 3′

• 3′ C T T A A G 5′

•  the EcoRI recognition sequence is

  • 5′ G A A T T C 3′

  • ____________ (fill in)

• Purify chromosomal DNA —>Digest with restriction endonuclease into many small fragments

• Purify the plasmid (the vector) —>Digest with restriction endonuclease that will create sticky ends that are compatible with the digestion products of the chromosomal DNA

first step of gene closning

Add DNA ligase to join them together

what do you do in gene cloning process once the chromosomal DNA fragments and digested vector are mixed together?

Introduce the recombinant vector with chromosomal DNA into host bacteria that are competent aka TRANSFORMATION

(all competent bacteria will uptake some digested DNA but only one bacterium will uptake the gene of interest, sometimes vector binds itself/returns to original structure/recircularized vector)

what do you do in gene cloning process once DNA ligase to joins sticky ends together?

• antibiotic resistance (like amp1 —> ampicillin resistance)

• •Vectors usually have a gene that confers….

die in the presence of the antibiotic like ampicillin

in the gene cloning process, cells that  did not get transformed do not have plasmid and will …

1. the lacZ gene to be disrupted

2. has a functional lacZ gene

1. in the recombinant vectors with chromosomal DNA, the vector was inserted in the region that contains lacZ gene which causes…

2. meanwhile the recircularized vector …

recircularized

the lacz protein product (Beta-galactosidase) produces blue colonies in the presence of X-Gal which means that bacterial colonies containing ___ vectors are blue

• •If it is, the colonies will be blue – no chromosomal DNA present

• •If not, the colonies will be white – chromosomal DNA present

 β-galactosidase is functional?

• •If it is, the colonies will be blue then ______

• •If not, the colonies will be white then_______

• •Bacterial host cell replicates the recombinant vector to produce many identical copies per cell; for example 50 copies

• •Bacterial cells divide around every 20 min giving rise to 10 million cells after overnight growth; 500 million copies of recombinant vector

• •Amplification of the DNA occurs in two ways:

complementary DNA (cDNA)

DNA that is made from RNA

false! could be single- or double-stranded

T/F: cDNA can only be double stranded

lacks introns

why is cDNA smaller in size than eukaryotic genes

only focusing on protein coding region

cDNA is Easier to clone if ….

cDNA

____ is required if expressing protein in bacteria which can’t splice out introns

PCR

Used to make large amounts of DNA in a defined region flanked by two oligonucleotide primers

Template DNA

Contains the region of DNA that needs to be amplified (PCR)

Oligonucleotide primers

Complementary to sequences at the ends of the DNA fragment to be amplified, These are synthetic and about 15–20 nucleotides long

true

T/F:  you need to know sequence of DNA to be bound by primers

•Deoxynucleoside triphosphates (dNTPs)

when template dna is mixed wtih primers it is also mixed with ____

• heating steps that inactivate most other DNA polymerases

• Thermostable DNA polymerase is necessary in PCR because PCR involves…

•Thermostable DNA polymerase

Taq polymerase obtained from Thermus aquaticus is an example of a ____

• •Denaturation – Template DNA strands separated by heating

• •Primer annealing – Primers bind to template DNA

•Primer extension – DNA polymerase copies the target DNA sequences by

•Three Steps of PCR

denaturation

PCR: Template DNA strands separated by heating

primer annealing

• Primers bind to template DNA

• Primer extension

– DNA polymerase copies the target DNA sequences by extending the primer in the 5′-3′ direction

thermocycler (•Automates the timing and temperature changes in each cycle)

PCR takes place in a machine called a _____

20 to 30

• •A typical PCR run involves __-__ cycles of replication

• •After 1 cycle, a DNA sample will increase 2-fold

• •After 20 cycles, a DNA sample will increase (~ 1 million fold)

• •After 30 cycles, a DNA sample will increase (~ 1 billion fold)

PCR:

• •After 1 cycle, a DNA sample will increase ____

• •After 20 cycles, a DNA sample will increase _____

• •After 30 cycles, a DNA sample will increase _____

Reproductive cloning

______ refers to methods that produce two or more genetically identical individuals

Somatic cells can give rise to whole new PLANT

(Somatic cells cannot give rise to whole new MAMMAL without special techniques)

why is cloning easier in plants?

1) mammary gland (2)electrical pulses (3) nucleus-free egg

for cloning Dolly: __(1)__ were collected and grown in the lab then __(2)_ were used until the initial cell was fused with a __(3)__ from another sheep —> zygote develops —> embryo implanted into surrogate

• •At 3 years, the length of her telomeres were like a 9- to 10-year-old sheep

• probably because dolly’s mother was 6 y/o so her telomeres were already shorter

• •Evidence suggested that Dolly may be “genetically older” than her actual age because ….

aberrant

cloning mammals may result in a shorter lifespan and ___ gene expression

agricultural yield; susceptible to rare diseases

• Livestock cloning may be advantageous with regard to _____

• However, such a herd may be more …

Stem cells

_____ supply the cells that construct our bodies from a fertilized egg and also replenish damaged cells in adults

1. Ability to divide

2. Can differentiate into one or more specialized cell types

Stem cells have two common characteristics

• Totipotent

____ stem cells can give rise to all cell types

•Pluripotent cells

______ (such as embryonic stem cells, ES cells) can differentiate into almost every cell, but can’t give rise to an entire, intact individual

pluripotent

• Embryonic germ cell in the gonads are _____

• •Multipotent cells

____ can differentiate into several cell types

• •Unipotent cells

____ can only differentiate into one cell type

1. basic genetic mechanisms

2. treat human diseases

Interest in stem cells centers around two main areas

1. They may help us understand the ______that underlie the process of development

2. They offer the potential to ______ or injuries that cause cell and tissue damage

false! Adult stem cells are rare and hard to isolate

T/F: adult stem cells are easy to isolate/commonly found

true!

T/F: Embryonic stem cells (ES cells) and embryonic germ cells (EG cells) can be grown in the laboratory

gene mutagenesis or gene editing

Experimentally altering the sequence of a gene is called _____ or _____

• CRISPR-Cas technology

_______ can be used to mutate genes in living cells

guide RNA (gRNA)

• •Researchers created a single RNA in which the tracrRNA and crRNA are linked to each other called ____

crRNA and cas 9 protein

tracrRNA binds to the transcribed product CRISPR locus (with phage DNA inserted in it) aka ____ and also ___

spacer

•The ______ region of the gRNA is complementary to one of the strands of the gene to be mutated

makes a double-strand break in this gene

once RNA binds to Cas9 and guides it to the gene of interest, Cas9 then …

• •Repair by nonhomologous end joining (NHEJ)

• Repair by homologous recombination repair (HRR)

• •After Cas9 cuts the DNA, two different repair events are possible
  

• •The region may incur a small deletion that inactivates gene

(after CRISPR makes double strand break) Repair by nonhomologous end joining (NHEJ) may cause…

• Repair by homologous recombination repair (HRR)

• (after CRISPR makes double strand break) when donor DNA homologous to the target region that also carries the desired mutation is added

• inactivate a gene

• create a point mutation

Use of CRISPR-Cas technology can eithe - or ____

• can be directly conducted on living cells

Major advantage of CRISPR over other methods is that it ….

1. Respect for autonomy:

2. •Beneficence

3. Non-maleficence:

4. justice

• •Four tenets of biomedical ethics

•Respect for autonomy

Consult and obtain agreement form people before conducting medical procedures on them

Beneficence

Patients to gain a net medical benefit

Non-maleficence:

There should be minimal harm

Justice:

Fair distribution of resources and respect for people’s right and morally acceptable laws

•Northern blotting

_____ is used to identify a specific RNA within a mixture of many RNA molecules

• •It can determine if a specific gene is transcribed in a particular cell type (nerve vs. muscle cells)

• •It can determine if a specific gene is transcribed at a particular stage of development (fetal vs. adult cells)

• •It can reveal if a pre-mRNA is alternatively spliced

what northern-blotting does:

• •It can determine if a specific gene is transcribed ________

• •It can determine if a specific gene is transcribed ________

• •It can reveal if …

• probed with labeled DNA fragment (put in solution containing probe DNA)

• RNAs that are complementary to the DNA probe are detected as labeled bands

in Northern blotting, RNA is extracted from cells and purified, electrophoresed (separated by size), and then …

Western blotting

_______ is used to identify a specific protein within a mixture of many protein molecules

• •It can determine if a specific protein is made in a particular cell type

  • •Red blood cells vs. brain cells

• •It can determine if a specific protein is made at a particular stage of development

  • •Fetal vs. adult cells

• •Western blotting has several uses

  • •It can determine if a specific protein is …

    • e.g.

  • •It can determine if a specific protein is ….

    • e.g.

• antibody

• labeled DNA strand

Western blotting uses a ____ as a probe and Northern blotting uses a ___ as a probe

epitopes

antibodies

antigens have ___ that are recognized by ___

epitoopes on antigens

antibodies bind to ____ on _____

primary antibody

in western blotting the ____ recognizes the protein of interest

primary antibodies usually lack labels/cant be detected so secondary antibody that recognizes the primary antibody is added as they carry enzyme that aids in creating a visible product

even though the primary antibody has recognized/bound to the protein of interest, ___ are added to…